Ballion B. (Poitiers, France)1, Belnoue L. (Poitiers, France)1,2, Brot S. (Poitiers, France)1, Gaillard A. (Poitiers, France)11Université de Poitiers, INSERM U1084, LNEC – Cellular therapies in brain disease -, Poitiers, France, 2CHU de Poitiers, Poitiers, France
Parkinson´s disease (PD) is mainly characterized by a progressive degeneration of mesencephalic dopaminergic (DA) neurons of the substancia nigra pars compacta (SNc). Loss of DA neurons induces major imbalances of basal ganglia loop activity resulting in motor and cognitive symptoms. Therapeutic proposals are multiple but among the most promising ones, the cell therapy is emerging as a major research axis. In this context, we propose to study the functional integration of DA progenitor neurons derived human induced-pluripotent stem cells (hiPSCs) grafted into the SN in the 6-OHDA lesioned RAG2-KO mouse model of PD.
In this project, we investigated the physiological functionality of the grafted-DA neurons, eight to nine months post-transplantation, by performing unit extracellular recordings in anesthetized mice to compare their electrophysiological characteristics to those collected in intact mice. Analysis of discharge activities (burst and irregular ones) and action potential (AP) shapes highlight that grafted-DA neurons show similar electrophysiological activities to that of native-DA neurons. These data point to an effective maturation of grafted DA neurons.
Because the striatum is the main target structure of nigral dopaminergic inputs, we aim to question in a second step, the restoration of the dorsolateral medium spiny neuron (MSNs) activities, as well spontaneous and cortical-evoked ones which are imbalanced in absence of DA in our PD model. We assume that dopamine release from grafted-DA neurons readjusts the MSN activity and for the purpose, our future experimental strategy is to set up optogenetics coupled with electrophysiological in order to light manipulate the grafted-DA neurons expressing photosensible ArchT channels and to observe the electrophysiological consequences on the MSN activity. For this purpose, we´re developing actually the combined approach setup using a model of C57BL/6JRj mouse in which an AAV-ArchT-GFP construct is injected in the SNc: our first results show that with a defined 550nm light stimulation pattern, we can inhibit transfected-AAV-ArchT-GFP DA neurons recorded spontaneous activity in anaesthetized mouse. This technical data is major for the study and will be apply to transplanted-DA RAG2-KO mice.
Belnoue L. (Poitiers, France)1,2,3, El Hajj H. (Poitiers, France)1,2, Bonnet M.-L. (Poitiers, France)1,2,3, Brot S. (Poitiers, France)1,2, Gaillard A. (Poitiers, France)1,21Inserm / U1084, Poitiers, France, 2Université de Poitiers, Poitiers, France, 3CHU de Poitiers, Poitiers, France
Traumatic brain injury (TBI) constitutes a leading public health concern, with limited treatment options available. Cell transplantation is a potential strategy to repair the injured brain. The recent discovery of human-induced pluripotent stem cells (hiPSC) offers an opportunity to generate patient-specific stem cells for cell-based replacement therapies. One highly clinically relevant cortical tissue in the context of cortical lesions is the motor cortex.
In this project, human IPSC were reprogrammed from dermal fibroblasts and then differentiated into three-dimensional aggregates of motor cortex progenitors. Our results showed at day 18 of differentiation, neurospheres displayed anterior fate, and still expressed neural stem cell marker (Nestin+). At day 46 of differentiation, cellular aggregates displayed motor cortical identity (CTIP2+, COUPTF1-), and expressed deep (CTIP2+, FoxP2+) and upper (CTIP2- and SATB2+) layers marker with layer V highly represented. In addition, neurospheres expressed neuronal (DCX, NeuN) and non-neuronal (GFAP+, Olig2+) markers.
In order to evaluate the therapeutic potential of motor cortical neurons derived hiPSC, D46-aggregates were grafted into the injured motor cortex. Two months after transplantation, the grafted cells expressed mainly the markers of immature neurons, and send projections to motor cortical targets not far from the transplant site such as the cortex and the striatum. Eight months after transplantation, the majority of grafted cells developed into mature neurons and expressed deep and upper cortical layer markers. Interestingly, the grafted neurons established specific point to point projections to the targets of motor cortex including the most distant targets such as the spinal cord. In addition, the grafted neurons established reciprocal synaptic connections with host neurons. Additional studies are underway to investigate the functionality of the transplanted neurons. These findings provide exciting evidences that hiPSC-derived motor cortical neurons could effectively re-establish the adult damaged motor cortical pathways.
Brot S. (Poitiers, France)1, Bonnet M.L. (Poitiers, France)2, Patrigeon M. (Poitiers, France)1, Francheteau M. (Poitiers, France)1, Belnoue L. (Poitiers, France)2, Gaillard A. (Poitiers, France)11Université de Poitiers / INSERM U1084, LNEC, Poitiers, France, 2CHU de Poitiers, LNEC, Poitiers, France
Parkinson’s disease (PD) is a neurodegenerative disorder associated with a progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compact (SNpc), leading to a loss of dopamine in the target brain region, the striatum. Cell replacement therapy in PD aim to restore the DA neurotransmission by transplanting new midbrain DA neurons precursors. Human induced pluripotent stem cells (hiPSC) is one of the most promising source of cells for autologous transplantation. Indeed, they are an ethical and unlimited source of transplantable cells. We used adult human dermal fibroblasts to reprogram to a pluripotent state, by means of mixture of mRNA reprogramming factors. The mRNA technology allows the generation of hiPSC under feeder-free conditions and eliminates the risk of genomic integration. After pluripotency characterization of hiPSC, we induced in vitro differentiation into dopaminergic neurons using a 3-step system. During differentiation, the cells were first induced into midbrain-specified floor plate progenitor cells, before expansion and differentiation into mature dopaminergic neurons. After 25 days of differentiation, the cells expressed DA precursor markers Nurr1 (38,7%) and FoxA2 (71%). After 51 days of differentiation, 43,6% of the cells are NeuN+. Among these neurons, around 70% are TH+. We grafted hiPSC derived DA precursors into the 6-OHDA lesioned SN in an animal model of PD. Eight months after transplantation, 46% of grafted cells are NeuN+ and 35,5% of neurons express DA marker TH. Among DA neurons, 46,3% are GIRK2+, a marker of SNpc DA neurons. Immunolabelling of hNCAM+ revealed graft-derived fibers along the nigrostriatal pathway into the medial forebrain bundle, and in the target structure of A9 DA neurons: the dorsal striatum. In addition, grafted animals showed a significant recovery in amphetamin-induced rotation from 3 months post-transplantation, and this effect persisted after 9 months. We have shown the possibility to obtain DA neurons of SNpc subtype from mRNA-reprogrammed hiPSC compatible with future clinical applications. More importantly, intranigral grafted neurons express midbrain specific DA markers and allow anatomical and functional reconstruction of degenerated nigrostriatal pathway in an animal model of PD.
Lainé A. (Poitiers, France)1, Brot S. (Poitiers, France)1, Rabesandratana O. (Poitiers, France)1, Gaillard A. (Poitiers, France)11LNEC – INSERM U1084 – Université de Poitiers, Poitiers, France
Traumatic brain injury is a critical public health and socioeconomic problem throughout the world, with limited treatment options available. We have previously reported that embryonic cortical neurons grafted into adult mouse motor cortex immediately after a cortical lesion allowed restoration of the damaged motor pathways. We have also shown that a delay between cortical lesion and cell transplantation can enhance graft vascularisation, survival and projections associated with better functional recovery.
To enhance graft survival, structural and functional recovery after injury, biomaterials protecting grafted cells and/or supporting repair processes such as extracellular matrix substitute are currently in development and could be a promising neurorestorative approach. Hyaluronan (HA) based hydrogels are receiving increased attention in tissue engineering because of their unique and appealing biological properties such as biocompatibility, biodegradability, and nontoxicity. Here, we investigated the therapeutic effect of HA-based hydrogel on host tissue after cortical traumatic injury. For this, we have implanted biomaterial into the motor cortex of adult mice 7 days after cortical lesion. Then, we have analysed its impacts on the secondary lesion, host cell migration, vascularisation of the biomaterial and inflammation of host tissue.
In the presence of hydrogel, we observed a reduction in lesion cavity volume compared with control. We have also observed the migration of SVZ-derived neuroblasts into the implanted biomaterial. These migrating neuroblasts are closely associated to blood vessels or with astrocytic processes. Interestingly, we observed formation of blood vessels in the implanted biomaterial. We have observed a reduction of glial scar around the lesion cavity. In addition, the biomaterial modulates macrophage polarization towards the pro-regenerative phenotype. Collectively these results suggest a beneficial effect of biomaterial after a traumatic cortical injury.
Rabesandratana O. (Poitiers, France)1, Lainé A. (Poitiers, France)1, Bonnet M.-L. (Poitiers, France)1, Belnoue L. (Poitiers, France)1, Brot S. (Poitiers, France)1, Gaillard A. (Poitiers, France)11Université de Poitiers, Equipe Afsaneh Gaillard, Poitiers, France
The cerebral cortex (CC) is the most complex structure in the mammalian brain, organized in six specific layers. The CC is the target of many pathological conditions such as Traumatic Brain Injuries (TBI). Cell transplantation is a potential strategy to repair the injured brain. One highly clinically relevant cortical tissue in the context of cortical lesions is the motor cortex, however to this date there is no reliable protocol to generate cortical neurons of motor identity. We have recently generated motor cortical neurons from human induced pluripotent stem cells (hiPSC). These neurons grafted into the injured motor cortex allowed reestablishment of the damaged motor cortical pathways.A major limiting factor after transplantation is cell death of the grafted neurons. To enhance graft survival, structural and functional recovery after TBI, biomaterials protecting grafted cells and/or supporting repair processes such as extracellular matrix substitutes are currently in development and could be a promising neurorestorative approach. To further improve in vivo grafts afterwards, motor cortical neurons as neurospheres were embedded with hyaluronic acid-based hydrogel complemented with heparin sulfate and denatured collagen, at day 18 of differentiation in vitro. Four weeks after embedding, neurospheres cultured either with or without hydrogel displayed similar spherical shapes and sizes in vitro. Numerous neurite projections were observed in both conditions, yet embedded neurospheres displayed three-dimensional neurite projections into the hydrogel, as opposed to neurospheres in control condition cultured onto an adherent coating. Embedded neurospheres showed longer-distance neurite outgrowth, and an important neurite fasciculation was observed, compared to neurospheres cultured without hydrogel. Using iDISCO clearing technique, specific cortical markers (CTIP2, COUPTF1 and FOXP2) showed higher levels of expression in the embedded neurospheres. Future experiments will consist in the grafting of cortical neurospheres encapsulated with optimized biomaterial into mice lesioned motor cortex to analyze in vivo the survival and the integration of the grafted neurons.
Patrigeon M. (Poitiers, France)1, Brot S. (Poitiers, France)1, Bonnet M.-L. (Poitiers, France)1, Belnoue L. (Poitiers, France)1, Gaillard A. (Poitiers, France)11Université de Poitiers / INSERM U1084, Laboratoire de Neurosciences Expérimentales et Cliniques, Poitiers, France
Parkinson´s disease (PD) is a neurodegenerative disorder characterized by resting tremors, rigidity, and hypokinesia. At the neuropathological level, PD is characterized by the degeneration of nigro-striatal dopaminergic (DA) neurons and by an abnormal accumulation of aggregated α-synuclein (α-syn), the main protein component of Lewy bodies. Intrastriatal transplantation of DA neurons from fetal ventral mesencephalic (VM) tissue in PD patients have provided proof-of-principle for the cell replacement strategy in this disorder. The grafted DA neurons can innervate the denervated striatum, improve striatal DA levels and in some patient lead to clinical improvements. However, following autopsy, 10-22 years after transplantation, some of the grafted neurons contained Lewy bodies similar to those observed in the host brain. The goal of our project is to investigate the propagation of α-syn from host to graft by comparing two transplantation sites, the substantia nigra (SN) and the striatum. Our hypothesis is that the striatum, which is not the natural site of DA neurons, represents an unfavorable environment for transplanted neurons, making DA neurons more vulnerable to the disease process. For this purpose, we set up a mouse model of PD by viral injection of AAV2-hα-synuclein A53T into the SNc of C57BL/6 WT mice. Four weeks post viral injection, we already observed a unilateral motor deficit in cylinder test. Our results show an accumulation of α-syn associated with a nigral DA degeneration from 6 weeks post viral injection up to 12 weeks. After model validation, DA progenitors from VM of β-actin GFP mouse embryo were grafted either in the SN or the striatum of lesioned mice. At different time points after grafting, we quantified the presence of α-syn in the graft. Our preliminary results, in both nigral and striatal grafts locations, show that the accumulation of human α-syn is higher at 5 month-post grafting compared to 1 month. Yet, from 2-month post transplantation, it appears that the amount of α-syn is higher in grafts located in the SN. This may be due to the greater viral load in the SN, which is the virus injection site. Ongoing experiments are underway to determine the impact of the viral injection site on host to graft α-syn propagation.
Thamrin N.P. (Poitiers, France)1, Bonnet M.L. (Poitiers, France)2, Patrigeon M. (Poitiers, France)1, Belnoue L. (Poitiers, France)2, Corvol J.C. (Paris, France)3, Corti O. (Paris, France)3, Brot S. (Poitiers, France)1, Gaillard A. (Poitiers, France)11Université de Poitiers / INSERM U1084, LNEC, Poitiers, France, 2CHU de Poitiers, LNEC, Poitiers, France, 3ICM, Institut du Cerveau et de la Moelle Epinière, Paris, France
Mutations in PRKN are the most common genetic cause for familial Parkinson’s disease (PD). The discovery of Induced pluripotent stem cells (iPSCs) makes it possible to generate patient-specific dopaminergic (DA) neurons to study how PRKN mutation causes PD. iPSCs can be differentiated into DA neurons, and be used as a human disease model for PD. However, one of the limitations is that culture models can typically last only several weeks not long enough to reproduce disease-specific phenotypes. One of the most important issues is the lack of in vivo cues necessary for the development and maturation of DA neurons. Some of these problems can be resolved by transplanting the cells derived-patient iPSCs to an animal brain. In this study, in a first step, we have investigated the impact of PRKN mutations in vitro on cell survival, differentiation, maturation and mitochondrial function of DA neurons derived patient with PRKN mutation and control subjects. After differentiation, more than 70% of the generated cells expressed DA precursor markers such as FOXA2, OTX2 & LMX1A in both subjects. However, there is an important reduction in the number of cells expressing mature DA markers in patient. The mitochondrial health has been analyzed by using Cytochrome-C antibody and a membrane potential marker: MitoTracker. At DA precursor stage, around 80% of mitochondria were metabolically actives in both subjects. However, at maturation stage, we observed a 15-25% decrease in mitochondrial activity in patient cells. Apoptosis detection using TUNEL or annexin-V assay showed 20% higher apoptosis in patient cells. Moreover, the mitophagy was 2-fold higher in patient compared to control. In a second step, we transplanted DA precursors derived patients and control into the substantia nigra in a mouse model of PD. One month after grafting, we found a 5-fold reduction in the number of DA mature neurons in grafts from patient cells compared to control. Interestingly, there was no difference between cell proliferation and neuronal maturation between both subjects. Experiments are underway to study the impact of PARK2 mutation on survival, axon elongation, pathogenic protein accumulation, cell-type-specific vulnerability and mitochondrial dysfunction of grafted DA neurons.
Plus d’informations sur le meeting ici : https://www.neurosciences.asso.fr/SN21/?page_id=153