The objective of the RESCUE project is to propose a new treatment for acute radiation syndrome (ARS) for the military personnel in operation who are victims of nuclear exposure, or for the public who are victims of malevolent acts. It is based on a breakthrough technology to produce hematopoietic stem cells (HSCs) capable of multi-lineage graft obtained from induced pluripotent stem cells reprogrammed from differentiated adult human cells. These original cells are called hiPSC (human induced Pluripotent Stem Cells). hIPSC have begun to revolutionize medical practices. The emergency treatment of populations requires the availability of ready-to-use frozen products to treat a large group of individuals. Drug" stem cell banks obtained from clinical grade hiPSCs will make it possible to produce stem cells of different types to treat the population. HiPSCs from these "universal" donors are already available in Europe. The RESCUE project aims to demonstrate that this strategy of using hiPSC to produce HSCs is the therapeutic response to the treatment of ARS. This project is in synergy with the new global strategies in terms of health and regenerative medicine, namely the creation of clinical grade hiPSC banks and the derivation of quality cellular products. The previous GIPSIS project (Astrid 2013-17), is a world first to demonstrate the generation of a long-term functional hematopoietic tissue from hiPSC. We have developed a cGMP (xeno-free) protocol for producing HSC from hiPSC. The reproducibility of this protocol has been demonstrated with 5 different hiPSC lines from 3 healthy donors and 2 leukemic donors. In addition, by selecting 2 surface markers, we are able to enrich our cell population containing HSCs capable of reconstituting functional hematopoiesis. These results are submitted to the prestigious journal Cell. The RESCUE project aims to demonstrate that this innovative therapy is applicable to a man developing an ARS. We will demonstrate this by validating this treatment in a "large" animal model developing hematopoietic syndrome. This will allow us to progress to a TRL of 6. In order to have a cell therapy product that can be used in clinics, we adopt the approach of validating a human HSC graft produced from hiPSC, frozen and ready to use, perfectly characterized and of a size that allows the treatment of a pig that has undergone exposure to whole body irradiation developing a reversible hematological syndrome of category H3 according to the MEdical TREatment ProtocOLs for Radiation Accident Victims (METROPOL) classification. We will demonstrate that human hematopoietic graft from hiPSCs is involved in hematopoietic reconstitution. Our strengths are our expertise in hematopoietic reconstitution, in the implementation of clinical trials (PHRC PRISME, NCT02814864), in the treatment of radiation victims and in the complementarity of a consortium with which we have been working for more than a decade. It is composed of academic researchers specializing in hematopoiesis, clinical hematologists, EFS Atlantic Bio GMP specializing in the production of advanced therapy medicinal products (ATMP) from from pluripotent stem cells and an SME; Phenocell, specialized in the production of clinical grade hiPSC banks. The RESCUE project is a demonstration that it will be possible in the future to treat victims of an ARS with freezable, ready-to-use products after nuclear exposure or malevolent acts. This will radically increase the capacity to manage and treat high-dose irradiated patients. The civil and military repercussions are immense. The shortage of organs and the rapid development of tissue bioengineering mean that regenerative medicine will have to use these techniques, particularly for the treatment of radiation sequelae.

De novo missense variants and inframe deletions were identified as causing neurodevelopmental disorders (NDD) characterized by intellectual disability, autism, brain anomalies and epilepsy in AGO1, AGO2 and more recently in AGO3. These genes encode Argonaute proteins which are involved in small non-coding RNA based gene-silencing pathways and have additional roles in regulation of transcription and splicing. The high percentage of sequence identity between AGO proteins and their target redundancy, as well as the strong overlap at the molecular (recurrent amino acid positions mutated in the different genes) and clinical (similar clinical manifestations) levels suggest that common mechanisms might be disrupted in NDD caused by AGO mutations. The objectives of PAgoDe are to dissect the clinical and molecular basis of these genetic disorders and how they overlap. To address these questions, we will first better delineate the clinical manifestations presented by individuals, identify novel variants in these genes and search for genotype-phenotype correlations. We will then combine approaches using complementary in vitro (human neural stem cell - hNSC) with in vivo (embryonic mouse cortices, drosophila) models to study the consequences of the patients’ variants and to decipher the respective roles of AGO proteins during brain development. We will analyze how expression of the two most recurrent variants on one hand and how knock-down of AGO genes (separately and simultaneously) on the other hand will affect miRNA functioning and mRNA regulation, but also neuronal differentiation and functioning. Finally, we will try to understand the phenotypic variability found for individuals mutated for the same gene and even the same variant, by searching for any correlation between the location of variants on protein (and their effect) and the clinical severity of the phenotype. We will also search, for individuals carrying the same variant, for potential second-hit or modifiers. In conclusion, this project will help to understand the roles of AGO proteins during brain development and how alterations of their functions can lead to neurodevelopmental disorder, which might lead to the identification of potential common therapeutic targets.

In all mammals, foetal cells are transferred during pregnancy to the mothers. These foetal microchimeric cells (FMC) (a) persist in low amounts in maternal bone marrow for decades after delivery, (b) are well tolerated by the maternal immune system, (c) harbor progenitor cells and (d) are able to migrate to damaged maternal organs where these adopt the phenotype of the lesioned tissue. Our group has demonstrated the constant recruitment of foetal cells in maternal wounds were these cells differentiate mainly as endothelial cells. We later identified a specific subpopulation of FMC expressing CD34+CD11b+CD31+ that peaks early post maternal wounding. These cells over express CCR2 opening the possibility to selectively recruit such foetal cells. Therefore, injections of low, physiological doses of Ccl2 leads to the rescue of delayed cutaneous healing in females previously pregnant, but never in virgin mice (or those pregnant with a CCR2 KO fetuses). In the skin granulation tissue, FMC form foetal-derived vessels and promote maternal angiogenesis through various peptides, mainly CXCL1. This project aims to further characterize FMC ability to help maternal repair as well as to obtain proof of concept of Ccl2 to rescue maternal healing in the human species. We aim therefore to: 1) Perform an in-depth characterization of the potentialities of FMC in mice. We will use lineage tracing and clonal analysis in a Confetti mouse model in order to investigate the heterogeneity and stemness of FMC residing in mothers as well as those specifically recruited to skin wounds. We will also perform a transcriptional profiling of these cells at single-cell resolution to decipher their hierarchy and diversity. 2) Demonstrate the therapeutic potential of FMC in maternal tissue repair in vivo. We will test whether CCL2 is an efficient and safe therapy to recruit FMC in Sickle Cell Disease and diabetic mice models that present impaired wound healing. Both types of wounds are severe and incurable. In addition, we will test whether low doses of Ccl2 can be beneficial in extra-cutaneous maternal tissue repair models, such as the brain with brain excitoxic injections. 3) Characterize CCR2 levels in FMC in pregnant women or after delivery. We will assess the expression of CCR2 (receptor for Ccl2 on FMC) in cord blood from pregnant women presenting or not chronic wounds. To identify FMC in post-parous women, we will isolate cells harboring paternal unshared HLA antigens. Then, we will measure the level of CCR2 mRNA of FMC in post-parous women with or without chronic wounds either linked to sickle cell disease or to diabetes. In case, there is no over expression of CCR2 in FMC from wounded women, we will perform RNA sequencing analysis of these cell populations in order to identify other signaling pathways involved in the recruitment of FMC after maternal wounding. This project will help develop an original strategy of a “natural stem cell therapy” by mobilizing FMC to promote maternal tissue repair during pregnancy and after delivery. Upon success, our study will allow a rapid translation of this “natural stem cell therapy” to the bedside.

Multipotent neural progenitors (NPs) give rise to fate-restricted neural cells either by successive cell divisions or by direct differentiation. The proteins that generate differentiated neurons from NPs are called “proneural proteins” (PPs). PPs are small transcription factors all belonging to the basic helix-loop-helix (bHLH) family and they come in three highly conserved flavours each with several sub-categories: Atonal homologues (ATHs), Achaete-Scute like (ASCLs) and Neurogenins (Neurog). PPs regulate both the proliferative capacity as well as the differentiation potential and daughter fates of NPs. How proneural proteins regulate these two aspects during neurogenesis is not fully understood. Evidence suggests that the oscillatory expression of proneural factors in NPs is an important feature of this regulation. A key pathway that cooperates closely with proneural proteins to regulate NP behaviour is the Notch cell-cell signalling pathway. Notch signalling ensures that NP decisions are communicated among neighbouring NPs and this communication is essential for ensuring the proper balance of progenitors and differentiated derivatives as development proceeds. Importantly, Notch signalling and PPs are tightly linked in a transcriptional negative feedback loop. Thus, NPs with high levels of Notch activity extinguish PP expression and retain NP fate, while cells with low Notch activity increase PP expression and generate neurons. Evidence suggests that Notch signalling exhibits more dynamic expression patterns than previously thought and that such dynamic expression is important for cell fate determination and morphogenesis. As a consequence of the Notch oscillations, proneural genes also express in an oscillatory manner. Although, this oscillatory expression of proneural genes seems to be mediated by a transcriptional mechanism, nothing is known about a possible post-transcriptional destabilization of proneural proteins. The dynamic expression of proneural genes driven by the oscillation of Notch activity is required in neural stem cell fate maintenance while in differentiating neurons, where Notch signalling is inactive, proneural genes expressed continuously and disappears when cell enter in terminal differentiation. Interestingly, the oscillatory dynamics of Notch signalling and proneural genes network is associated with the proliferation of progenitor cells that sequentially give rise to different types of cells by changing their differentiation competency. What drives these changes in cell competence upon Notch/proneural oscillation, remains an open question. Post-translational regulation of PPs has emerged as a key regulator of their dynamics with potentially important implications for NP cell fate. In particular, phosphorylation of a Serine/Threonine residue present in all PPs regardless of species or sub family is essential for regulating their expression and activity. It remains unclear however how post-translational regulation influences NP behaviour in different developmental and evolutionary contexts; whether and how Notch signalling cross-talks with post-translational PP modifications and how NPs implement a cellular memory of such interactions to maintain an NP versus a differentiated state. Finally, an increasing number of studies highlight the importance of chromatin remodeling in progenitor fate maintenance as well in the restriction of developmental competence during cell differentiation. How changes in Notch dynamics and proneural protein phosphorylation cell autonomously regulate chromatin states is unclear. This project will address this knowledge gap through five conceptually linked but experimentally independent objectives using an innovative, cross-species comparative approach to neurogenesis.