Researcher: Associate Professor Anthony White
We have made good progress with the project funded by the Col Bambrick Memorial MND Research Grant. Our overall aim is to generate cell models of the brain that include ALS patient microglia, which are the brain’s main immune cell. We plan to do this using two different approaches both of which have their advantages and disadvantages. The first aim is to generate microglia from blood derived immune cells called monocytes. This allows rapid generation of microglia-like cells from a larger number of patients, but these cells are microglia-like rather than being ‘true’ microglia. The second aim is seeking to generate ‘true’ microglia from patient stem cells. While these cells are perhaps a better model of real microglia, they are also harder and more time consuming to generate and it is not feasible to generate microglia from large numbers of patients. Instead these are used to understand immune cell processes in ALS rather than use for initial testing of new drugs. In both aims we want to examine how the microglia function in a model of the brain, hence we are seeking to generate a 3D model including ALS patient neurons, astrocytes (a cell type important for maintaining healthy neuronal function) and the microglia.
For our first aim, we initially projected that we would receive blood samples to generate monocyte-derived microglia from 6 ALS and 6 control cases. We have now received a total of 23 blood samples from our collaborator in Italy including 6 with rapidly progressing ALS, 5 with intermediate progression and 5 with slow progressing disease plus 7 matched controls. We have so far converted 2 rapid, 1 intermediate and 1 slow case as well as 1 control into microglia. We have found that microglia from people with rapid disease seem to form microglia-like cells faster than cells from age-matched and younger controls. We are currently investigating the cells further to try and understand why this is occurring and what it means for microglia-mediated inflammation in ALS.
We have also demonstrated feasibility of using the monocyte-derived microglia to screen for patient-specific drugs. We have developed a model system using a high throughput 96 well plate to screen for compounds that may improve the function of microglia on a patient by patient basis. We are starting to treat some of the ALS and control microglia with in-house compounds that control inflammation, to see how the cells respond. We will know the outcomes of these assays soon. We are now planning on developing this further to determine if we can identify patient-specific beneficial drugs.
To generate the stem cell lines for both aims, we have 2 x disease onset (C9orf72 and sporadic), 2 x healthy carriers (C9orf72 and FUS P525L), and 2 x controls fibroblast samples that have been received from our Italian collaborator. All these fibroblasts have been re-programmed from skin fibroblasts into stem cells and ‘banked’ for further use. We have fully developed our ‘true’ microglia generation method and characterised the cells to determine that they express a range of processes expected of ‘true’ microglia. We are now about to select the best cell lines to generate motor neurons and astrocytes to co-culture with microglia in 3D models.
We are also about to use these models to try and understand further why some people with ALS progress very quickly while others (sometimes with the same mutation) progress more slowly. We have ideal tools now to start to understand this.