Putting the Patient First: Answering the big questions with patient-derived cell lines

Author: Gráinne Geoghegan

Academic Unit of Neurology, Trinity Biomedical Sciences Institute, Trinity College Dublin

One of the most frustrating aspects of a diagnosis of Motor Neuron Disease (MND) or Amyotrophic Lateral Sclerosis (ALS) is the lack of definitive answers to the big questions. ‘Why did this happen to me?’ ‘Could I have prevented this had I known earlier? ’‘What can be done about it?’. For scientists and clinicians, finding the right answers to these questions can sometimes feel like finding the needle in a haystack. 90% of ALS/MND cases occur sporadically, meaning that there is no clear underlying cause for disease onset, although there are likely a variety of different genetic and environmental risk factors involved (1). So without knowing the answer to ‘why did this happen’, it’s very difficult to develop the treatment options to ‘do something about it’.

Barriers to New Therapies

Research is ongoing to try to more conclusively determine the underlying causes and risk factors for developing ALS/MND in order to improve diagnoses and develop new treatment options. However, a significant stumbling block for new therapies is the lack of accurate disease models available to scientists to test their hypotheses.

A ‘disease model’ is a laboratory representation of some, or most aspects of a particular disease. These models can be anything from special cells in a dish, to animal models like mice, fish, flies and worms whose genome has been altered to express multiple copies of specific genes responsible for some aspects of the disease, or even organotypic cultures, which are pieces of tissue taken from patients or animals and kept alive in nutrient-rich media. Although all of these types of disease models have helped improve our understanding of ALS/MND, it is important to note that even the best model can only mimic certain aspects of the disease, and none can truly encapsulate everything going on at both cellular and behavioural levels.

Moreover, ALS/MND is very difficult to model in a laboratory environment because it is such a complex disease. Great progress has been made in identifying genetic causes for ALS/MND in many different types of animal models like zebrafish, rats and mice, and in cell-based models. However, all of these models have limitations as they are based on manipulating the genome of these animals in order to get them to display symptoms of a disease which they do not get naturally. Furthermore, these models are based on the inherited form of ALS/MND, which only accounts for approximately 10% of cases (1).

Patient-Focused Solutions

To overcome some of these limitations, researchers have developed new, more specific models of ALS/MND based on patients themselves. Using advanced cell reprogramming technologies, scientists can take a small skin biopsy from a patient and grow skin cells called fibroblasts from it in the lab. These cells can then be reprogrammed to become neurons (outlined in Figure 1), or other cell types found in the brain which are also involved in ALS/MND disease progression (2). These unique cells retain the genetic make-up of the patient, making them a highly specialised model that more closely resembles human disease and can mimic both inherited and sporadic forms of ALS/MND. Furthermore, as ageing is the biggest risk factor for all neurodegenerative diseases, advances in this technology has allowed these cells to retain their ageing signature (2). Scientists can then test their hypotheses using this model, which produces more accurate findings.

Figure 1. Overview of how skin biopsy samples are used to make induced pluripotent stem cells (iPSCs), which can be used as a valuable drug screening tool.

This technology is based on the ground-breaking development of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka, for which he was awarded the 2012 Nobel Prize in Physiology or Medicine. Using this technology, fibroblasts can be genetically reprogrammed to become pluripotent stem cells, which are a type of unspecialised cell capable of giving rise to all the cell types of the body when grown under the right conditions. This pioneering technology overcomes the highly contentious issues surrounding embryonic stem cells in research and allows for the development of patient-specific cell lines (population of cells with the same genetic make-up) from a simple skin biopsy.

Promising Discoveries

iPSC technology was first used to grow iPSC-derived motor neurons from patient samples in 2008 by Dimos and colleagues, and since then, many pioneering discoveries and breakthroughs have been made (3). iPSC-derived motor neurons were used to confirm the importance of other cell types in the brain for disease progression, an important discovery which could lead to the development of new targets for therapy (4). In 2014, three separate studies using iPSC-derived motor neurons identified shared molecular pathways leading to ALS/MND which were common to patients with different genetic backgrounds (5). This means that patients with different genetic mutations known to cause ALS/MND, for example C9orf72 and SOD1, could benefit from similar therapies, if the underlying pathway to disease is the same.

Furthermore, many potential new drug candidates for ALS/MND have been identified using patient-specific, iPSC-derived cells. Three drugs, ropinirole (6), retigabine (7) and bosutinib (8) were identified as potential new therapies for ALS/MND in drug screenings using iPSC-derived motor neurons, all of which were then tested in clinical trials. All three drugs are already approved and used in the treatment of other conditions, namely Parkinson’s disease, epilepsy and chronic myeloid leukaemia, respectively. This illustrates that iPSC-derived motor neurons could be used to test drugs which are already approved and repurpose them for the treatment of ALS/MND, dramatically shortening the time it would take for patients to gain access to new therapies.

These developments have also opened the door to the possibility of personalised medicine in ALS/MND. As iPSC-derived motor neurons retain the genetic signature of the donor patient, it’s possible to identify potential new therapies which will be more efficacious for certain subgroups of patients, based on the genes these patients express. Furthermore, it is conceivable that patients could give a skin biopsy sample from which iPSC-derived motor neurons are grown, and are then used to predict that patient’s specific response to treatment. This would help to ensure that the right drug is given to the right patient, not only leading to better treatment outcomes, but also reducing unwanted side effects.

iPSC-derived motor neurons are already helping us to answer some of the big questions in ALS/MND research, from identifying underlying causes and developing new biomarkers, to discovering new therapies. The use of iPSCs has exploded in popularity since their development just 15 years ago for a good reason. Scientists conduct their research to better understand the human body and how to treat it when things go wrong. Therefore, when the patients themselves are put at the forefront of this great effort, more accurate findings and bigger breakthroughs are made.

Our Work in Trinity College Dublin

Professor Orla Hardiman’s group in Trinity College Dublin and Beaumont Hospital is setting up a new biobank of skin cells with funding from FutureNeuro, the Science Foundation Ireland Research Centre for Chronic and Rare Neurological diseases, and in collaboration with researchers in NUIG, RCSI and the University of Sheffield. A small piece of skin, smaller than the top of a pencil (4 mm) is taken from a numbed area on the inner forearm using a circular ‘punch’ blade and heals without a scar. The biopsy is then taken to the lab where researchers grow skin cells from it, which are then ready to be stored in the biobank. This valuable resource will be used to investigate the underlying causes of ALS/MND and to develop and test new treatment options in the future.

We are very grateful to everyone who has taken the time to donate a sample of any kind so far. If you or your family members are interested in donating a sample to the biobank, or would like more information, contact Mark Heverin, Research Manager to Prof. Hardiman (mark.heverin@tcd.ie) or Gráinne Geoghegan, Research Assistant (grainne.geoghegan@tcd.ie).

References and Further Information:

1. Hardiman O, Al-Chalabi A, Chio A, Corr E, Logroscino G, Robberecht W et al. Amyotrophic lateral sclerosis. Nature Reviews Disease Primers. 2017;3(1).
2. Myszczynska M, Ferraiuolo L. New In Vitro Models to Study Amyotrophic Lateral Sclerosis. Brain Pathology. 2016;26(2):258-265.
3. Dimos J, Rodolfa K, Niakan K, Weisenthal L, Mitsumoto H, Chung W et al. Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons. 2008;321(5893):1218-1221.
4. Meyer K, Ferraiuolo L, Miranda C, Likhite S, McElroy S, Renusch S et al. Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS. Proceedings of the National Academy of Sciences. 2013;111(2):829-832.
5. Matus S, Medinas D, Hetz C. Common Ground: Stem Cell Approaches Find Shared Pathways Underlying ALS. Cell Stem Cell. 2014;14(6):697-699.
6. Fujimori K, Ishikawa M, Otomo A, Atsuta N, Nakamura R, Akiyama T et al. Modelling sporadic ALS in iPSC-derived motor neurons identifies a potential therapeutic agent. Nature Medicine. 2018;24(10):1579-1589.
7. Wainger B, Kiskinis E, Mellin C, Wiskow O, Han S, Sandoe J et al. Intrinsic Membrane Hyperexcitability of Amyotrophic Lateral Sclerosis Patient-Derived Motor Neurons. Cell Reports. 2014;7(1):1-11.
8. Imamura K, Izumi Y, Watanabe A, Tsukita K, Woltjen K, Yamamoto T et al. The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis. Science Translational Medicine. 2017;9(391):eaaf3962.