The Use of Stem Cells in Motor Neurone Disease (MND)

Motor neurone disease (MND) or amyotrophic lateral sclerosis (ALS) is a fatal late onset neurodegenerative disorder that results in progressive and selective degeneration of motor neurones (MNs) through poorly understood mechanisms [1,2]. At present there is a lack of therapies that can significantly alter the course of ALS, and usually death occurs within three years from symptom onset due to respiratory failure.

MND is a relatively rare neurodegenerative condition with an annual incidence of 2.0-2.5 per 100,000 in the European population. The majority of cases are sporadic (sALS), but 5-10% of all cases present an inherited form of the disease (familial ALS, fALS). Following the discovery in 1993 that SOD1 mutations are associated with fALS3 a growing number of genes have been associated with inherited forms of this disease. One of the most significant is TARDBP, which encodes the protein TDP-43. This protein is involved in splicing regulation and mRNA biogenesis and has been recently shown to be a key protein in MND. TDP43 related pathology is present in the vast majority of ALS cases [4]: with the exception of SOD1 and FUS related fALS, all other cases of this disease present mislocalisation and accumulations of ubiquitinated TDP-43.

Studying familial ALS particularly SOD1 mutations has been highly influential to our understanding of disease pathogenesis. One such insight has been the recognition of the importance of noncell autonomous mechanisms of neurodegeneration. Specifically, the glial environment (in particular astrocytes and microglia) is thought to be central to disease progression [5]. In turn, this has led conceptually to novel approaches to neuroprotection, including cell based approaches to manipulate the ‘hostile’ glial environment.

Against this background stem cell based approaches, focusing on forms of fALS, are increasingly attractive as a platform for developing, both models and potential treatments for MND.

Stem cells

Stem cells are found in all multicellular organisms and can be defined by two key properties: 1. the ability to self renew, and 2. the ability to differentiate into a range of specialised cell types according to their potency. Potency ranges from pluripotent stem cells, such as embryonic stem cells, that are able to generate every cell type in the organism to multipotent tissue specific stem cells that are able to generate those cell types that make up the tissue of origin. For example, neural stem cells can generate the neurones, astrocytes and myelinating cells that form our nervous system.

Since the discovery of reprogramming technologies in 2007 [6], it is also possible to generate artificially induced pluripotent cells (iPS) from adult cells, for example skin fibroblasts. The basic idea behind this advance is the introduction of two to four key reprogramming’ factors to ‘reset’ the cellular machinery from an adult cell to an embryonic-like pluripotent cell. The practical implication of this technology is the increasingly widespread availability of patient specific stem cells for research or potentially treatment.

Combining these ideas with advances in human stem cell biology and new methods to generate region specific functional neurones, including MNs, and glial cells offers much promise for regenerative neurology. The absence of treatments and the appalling prognosis of MND has led to MND being considered an appropriate candidate disease for innovative experimental medicine. One of these approaches is the use of stem cells.

Using stem cells to treat MND

Conceptually cell based therapies can be categorised based on the putative mechanism of efficacy. In broad terms this can be 1. cell replacement and 2. using stem cell not to replace cells or tissue but rather to manipulate the environment around the injured or dying neurones (Figure 1).

Cell replacement, conceptually straight-forward, seeks to replace dying or lost cells with ex vivo generated cell specific progenitor(s). Aside from the challenges of immune-rejection, the basic biology to restore connectivity in the context of an adult and scarred environment remains poorly understood. In contrast to Parkinson’s disease, where the key cell loss is specific to a restricted area in the midbrain, loss of MNs is extensive, ranging from cortical upper MNs to lower MNs: for this reason a successful replacement therapy would require transplanted MN progenitors to integrate into a pre-existing network. Although it is now possible to generate functional motor neurones by using protocols based on neurodevelopmental insights [7,8] for the reasons discussed above it is presently unrealistic to use stem cells to replace MNs in MND patients. Notwithstanding this major challenge to clinical human application, it is of interest that experimental studies involving transplantation of stem cells or differentiating MNs into animal models of MND have been reported. These studies have been valuable in understanding the potential of stem cell transplantation in MND, and viewed collectively some general conclusions can be drawn: 1. the majority of studies use models of SOD1 fALS which might not necessarily be the most relevant form of the disease in humans; 2. in many cases the onset of the
disease and/or the symptoms observed in the animal models is different from that observed in humans [9] ; 3. stem cell transplantation is often performed before the manifestation of clinical signs, which is at variance with the clinical scenario; 4. in those stem cell transplantation studies in which amelioration of the phenotype was observed, there is little evidence that this effect was due to replacement of lost MNs in the pre-existing neuronal network [10]; 5. increasing evidence suggests that the functional recovery observed is most likely a consequence of stem cells modifying and thus exerting a neuroprotective effect.

This indirect non-cell [MNs] replacement neuroprotective effect of cell transplantation is of growing interest: even if stem cell therapies are not at the moment able to achieve replacement of the lost neurones, the possibility to exploit different mechanisms to slow disease progression might represent a viable therapeutic approach. The precise mechanism of cell mediated neuroprotection is unclear. Implicated mechanisms include cell based secretion of trophic factors and immune modulation. Several studies highlight this beneficial bystander effect: for example, Vercelli and coworkers [11] used human bone marrow stromal cells (hMSCs) in a SOD1 model, observing no new MNs after transplantation, but a 35% protection of endogenous MNs. In a different study, Lepore and colleagues [12] demonstrated functional improvement in a similar model following focal transplantation of glial progenitors. In both these studies the actual mechanism of neuroprotection is unknown although some evidence suggests production of trophic factors by transplanted cells may be important. Indirect evidence supporting the importance of neurotrophic factors in a study of transplantation of stem cells engineered to secrete neurotrophic factors is beneficial in a model of MND [13].

Against this background human studies of stem cell transplantation are increasingly being proposed. Indeed one early clinical phase study is ongoing, studying the effect of foetal neural stem cell transplantation, targeting the cervical spinal cord and using respiratory measures as a secondary outcome, with safety being the primary aim. This study is based at Emory University [14].

Stem cells as a model of MND

Although stem cells for transplantation tend to capture the headlines it is likely that the major benefits of stem cells will actually emerge from in vitro experimental studies that ultimately lead to discovery of new therapeutics. Despite major advances in our understanding of MND, the actual mechanism of MN loss remains unknown. Therefore even in the presence of a successful method to reintegrate functional MNs to regain motor function or modification of the environment to slow progression there is a clear need for further disease models to understand better disease causation.

Notwithstanding the valuable insight from animal models there are several limitations for direct human extrapolation [9]. These include: interspecies differences ranging from differences in the physiology to species specific gene splicing patterns [15] (particularly important considering for example the prominent role of TDP-43 in splicing regulation [16]), disproportionate emphasis on SOD1 based models, and the fact that the techniques used to generate animal models present intrinsic limits to translation. Specifically, the majority of animal models for MND over-express either the wild type or mutant form of the human gene which results in some cases in premature or juvenile rather than age related neurodegeneration. Using human stem cells to model disease can overcome some of these issues. For example generating iPS disease lines from familial patients allows the study of mutant proteins in a physiologically relevant context. There are several examples of succesful application of using iPS to model neurological and brain disorders such as spinal muscular atrophy (SMA)17, inherited Parkinson’s disease18 and schizophrenia [19].

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These systems can also be reduced to high throughput assays and as such are a potentially powerful platform for efficient drug discovery. In summary human stem cell biology and its applications offer new approaches to study and potentially treating MND [l].

References

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