| 1 | Nuffield Department of Clinical Neurosciences | Generating human sensory neurons from induced pluripotent stem cells | 1495 | 15 | 1 | 45.1 | neutral | 5:20 | I'm just going to introduce our project in which we've generated human centrenure on from induced pro-apotent stem cells in order to generate a malinating co-culture system. And the reason we wanted to do that is it's really essential for healthy peripheral nerve function a very close and intimate relationship between axles and shwan cells. So axles are malinated by shwan cells and there's complex bidirectional signaling between these two different cell types. Now that signaling can go wrong in disease states such as inherited neuropathy, inflammatory neuropathies and we obviously want to understand that better, preferably using a humanized system in which we have the native proteins in their normal environment. And so to do that we've taken advantage of really this emerging technology which is induced pro-apotent stem cells. The principle there is we will take a skin barbeque from a patient, generate fiber blasts and then we can reprogram those fiber blasts with a number of oncogenic factors. We can then generate pro-apotent stem cells that can give rise to any cell type in the body but in this case we've differentiated them into sensory neurons. And then the aim of this project is to combine these sensory neurons with rodent shwan cells and really to understand if we can generate long term malinating cultures using this system and most importantly to see if we can use it as a model system to study disease. In order to establish the malinating co-cultures we first differentiate the IPS cells to sensory neurons, mature them for several weeks and then seed in the primary shwan cells. The subsequent alignment and onset of malination recapitulate exactly those same events that occur in vivo. So here you're seeing a live malinating co-culture. You can see the individual human cell bodies and the phased bright elongated structures are the malinated axons. We've performed cross-sectional electron microscopy imaging through these maline segments and you can clearly see the individual maline wraps and the surrounding basal lamina. Between each maline internode we see successful formation of the node of rombiae with correct compartmentalization of key nodal proteins including fortish-gated sodium channels at the node and contactin associated protein in the adjacent paranode. The shwan cell and axon are in very close contact and are constantly signalling to each other. One crucial pathway vital in the initiation and maintenance of malination is the nureglin a B receptor pathway. We've overexpressed nureglin 1 type 3 specifically in the sensory neurons using AAVs and we see a dose dependent increase in the levels of maline production. So this confirms that this vital pathway is active in our malinating co-cultures. So we've shown that we can successfully model the interactions between a human sensory neuron and a shwan cell in vitro. So the next step is to investigate a pathological scenario that impacts on the functioning of these two cell types. The development of a culture system involving the malination of human sensory neurons provided us with an viable opportunity to investigate the pathology of immune-mediated demanding nurepathies. In this study we primarily focus on the chronic condition chronic etaxic nurepathy with optalmplegia, m-protein, cold occluutins and anti-diysylosylantoblase, also known as canemate. The major reason for this focus is that this nurepathy has prominent and often disabling sensory dysfunction as Tony Morris began to experience symptoms of this disorder in the early 1980s, other struts. In the early 80s I started to feel my feet were very uncomfortable and I was generally walking very cumtsyly. A few years later it the numbness and the lack of control moved at my legs. This meant I didn't really have any balance so I couldn't stand or walk and whilst initially things were very uncomfortable and there was a lot of pain and discomfort. Once the peripheral nurepathy had spread throughout my entire body I lost all sensation of pain. The whole of my body had lost its sensation. By applying anti-diysylosylantoblase to our malignant co-culture system we're able to demonstrate both the topographical targets of these antibodies in culture and also their pathological effects. Antibodies were found to target the exposed nodal and unmalinated axolema as shown here. With the addition of a source of complement acute and extensive axonal degeneration ensued. However canemate has demilinating as well as axonal features. We speculated that if demilination was also mediated by anti-diysylosylantoblase then this bit may be via a more chronic complement independent mechanism. The impressive longevity of our cultures allowed us to continuously apply diysylosylantoblase over two four week periods both in the onset of malination and also in established six to nine month old cultures. These experiments revealed that this exposure both prevented malination and also induced demilination without affecting axonal integrity. This work is therefore established that anti-diysylosylantoblase have pleiotropic pathological effects. Co-cultures now provide an ideal system in which to further establish the mechanism of these pathological effects and also to test new therapeutic strategies. | ↗ |