Camille Thiberge

Maria Llach Pou

Aline Vitrac

Uwe Maskos

Isabelle Cloëz-Tayarani
email: isabelle.cloez-tayarani@pasteur.fr

Humanized Chimeric Mouse Models to Study Human Neural Development and Pathogenesis of Brain Diseases

Keywords: Xenografting, in utero transplant, human stem cells, induced pluripotent stem cells, chimeric mouse models, neurodevelopment, neurodevelopmental disorders Humanized mouse models and neural development

The development of humanized neural chimeric mouse models consists in grafting human neuronal progenitor cells (NPC) or differentiated neurons derived from induced pluripotent stem cells (iPSC) into the mouse brain at different timepoints. As the main features of cortical networks and their alterations during brain development cannot be reproduced in vitro, these in vivo models have been used to investigate the mechanisms by which reprogrammed human neurons and non-neuronal cells integrate and migrate into the mouse brain at early stages of brain development and display functional activities several months after their grafting. Here, we describe the neonatal grafting technique of human NPC which we use in our laboratory.

We also present a new method based on the grafting of human NPC into the brain of mouse embryos, in utero. A third method consists in the stereotaxic grafting of human NPC into selected brain regions of adult mice. The iPSC technology combined with the use of chimeric mouse models offers numerous possibilities to study human neural development within wellcontrolled and defined temporal windows and to model neuropathological disorders.

Introduction

The reprogramming of human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) into neuronal progenitor cells (NPC) and mature neurons is of major interest to study brain development during the critical periods of establishing diversity, connectivity, and functional maturation of neurons within the central nervous system (CNS). These properties are particularly important within the cortical regions and the targeted areas of their innervation. A specificity of the cerebral cortex resides in the existence of multiple cortical sections that are structurally and functionally organized into connected units to exert different functions through a large variety of neuronal subtypes [START_REF] Barbas | General cortical and special prefrontal connections: Principles from Structure to Function[END_REF]. At later stages of development, post-mitotic neocortical neurons conserve a high degree of plasticity which allows the postnatal reorganization of specific neural microcircuits in vivo [START_REF] De La Rossa | In vivo reprogramming of circuit connectivity in postmitotic neocortical neurons[END_REF] .

To date, different ESC and iPSC reprogramming protocols have been developed which can be used to produce various neuronal subtypes [START_REF] Corti | Human pluripotent stem cells as tools for neurodegenerative and neurodevelopmental disease modeling and drug discovery[END_REF] . In particular, the reprogramming of stem cells into human cortical neurons has made it possible to study various developmental features after their grafting within the prefrontal cortex [START_REF] Espuny-Camacho | Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo[END_REF] . Interestingly enough, it has been shown that grafted ESC-derived human cortical neurons can integrate functionally into the lesioned mouse brain, when their cellular identity corresponds to that of the brain area under grafting [START_REF] Espuny-Camacho | Human Pluripotent Stem-Cell-Derived Cortical Neurons Integrate Functionally into the Lesioned Adult Murine Visual Cortex in an Area-Specific Way[END_REF]. In humans, the cortex is organized into six layers [START_REF] Amunts | Architectonic mapping of the human brain beyond Brodmann[END_REF] which contain neurons with selective spatial organization and distinct biochemical, morphological, and electrophysiological characteristics [START_REF] Molyneaux | Neuronal subtype specification in the cerebral cortex[END_REF][START_REF] Jiang | The organization of two new cortical interneuronal circuits[END_REF][START_REF] Lake | Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain[END_REF][START_REF] Martinez | Anatomical and electrophysiological clustering of superficial medial entorhinal cortex interneurons[END_REF] . The axonal projections of these cells are also specific not only to their cortical location, but also to the neuronal phenotype. Thus, the excitatory pyramidal cortical neurons project into various but specific regions of the brain while the interneurons' projections remain local within the cortical layers.

Xenografting of human neuronal progenitor cells into a rodent host allows their study in vivo on an extended time scale (up to 10-12 months) [START_REF] Linaro | Xenotransplanted human cortical neurons reveal species-specific development and functional integration into mouse visual circuits[END_REF][START_REF] D'alessio | Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex[END_REF] as compared to in vitro studies (70-80 days in culture) [START_REF] Gouder | Three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells[END_REF]. In addition to such an important temporal aspect, the growth of human xenografted neurons takes place in a vascularized environment, which is essential for the survival of the transplanted cells. It has also been shown that the oligodendrocytes of host mice have the ability to myelinate the axons of grafted human neurons [START_REF] Espuny-Camacho | Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo[END_REF], which is an essential process to their long-term functional activity. The cortical identity of the grafted neurons, which depends on the layer in which they are positioned, as well as the temporal characteristics of their development are also maintained after transplantation [START_REF] Espuny-Camacho | Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo[END_REF][START_REF] Linaro | Xenotransplanted human cortical neurons reveal species-specific development and functional integration into mouse visual circuits[END_REF][START_REF] Real | In vivo modeling of human neuron dynamics and Down syndrome[END_REF].

Thus, the maturation of human neurons can take place over several months despite their development in a more mature murine environment [START_REF] Linaro | Xenotransplanted human cortical neurons reveal species-specific development and functional integration into mouse visual circuits[END_REF]. A recent study has shown that when NPC are transplanted together with deep-layer cortical neurons into the brain of adult mice, the first synapses are visible after only 20 days of development and their number increases gradually overtime [START_REF] Real | In vivo modeling of human neuron dynamics and Down syndrome[END_REF].

A summary of the xenografting studies carried out to date with human cells is presented in Table 1. It should be noted that most of these studies have been performed in newborn or adult rodents with ES-or iPS-derived human neurons. The main phenotypes studied are also indicated, whether they are morphological and / or functional.

Here, we first describe the step-by-step protocol used for neonatal transplants. This protocol has provided the means to analyze some of the processes which take place during the early stages of neuronal maturation, and on the role of SHANK3, a postsynaptic protein which is altered in autism spectrum disorders (ASD) as we reported previously [START_REF] Vitrac | A chimeric mouse model to study human iPSCderived neurons: the case of a truncating SHANK3 mutation[END_REF]. In the course of this study, we also found that the maturation of the same reprogrammed neurons was slower in vivo as compared to in vitro conditions, using the same cells in both studies [START_REF] Vitrac | A chimeric mouse model to study human iPSCderived neurons: the case of a truncating SHANK3 mutation[END_REF][START_REF] Gouder L | Altered spinogenesis in iPSC-derived cortical neurons from patients with autism carrying de novo SHANK3 mutations[END_REF] .

We will then describe a step-by-step protocol which consists in grafting human-derived NPC in mice in utero. This new method, which has not been used in mice to date, provides the means to study the early stages of human development and neural precursors events before they start differentiating. Another important aspect resides in the fact that in utero grafting allows the use of immunocompetent mice. Finally, we describe a transplantation method in adult mice which can be particularly adapted to the grafting of inhibitory neurons which have limited projections to cortical areas. We will highlight the diverse applications of each of these methods for studying different pathologies at their earliest stages during brain development.

Materials

Media

-NPC culture medium: The culture medium consisted of 500 ml of DMEM/F-12 mixed with 500 -The following is the list of primary antibodies and their corresponding secondary antibodies:

Methods

Neuronal progenitor cells (NPC) from human iPSC lines

The purpose of this section is to specifically describe the protocols which allow amplification and labeling of neuronal progenitor cells (NPC) before their grafting into the mouse brain.

For this study, we used two control cell lines from Coriell Biorepository (Coriell Institute for Medical Research), the GM04603 (https://www.coriell.org/Search?q=GM04603) and GM01869 (https://www.coriell.org/Search?q=GM01869). Their derivation into NPC and their characterization have been described elsewhere [START_REF] Boissart | Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening[END_REF]. For protocol optimization we used the WTSli002-A line (https://cells.ebisc.org/WTSIi002-A/ Wellcome Trust, UK) as in a previous study [START_REF] Mouilleau | Dynamic extrinsic pacing of the HOX clock in human axial progenitors controls motor neuron subtype specification[END_REF]. Protocol modifications result from the constant development of new commercial products to simplify and optimize iPSC-derivation protocols, without altering the cell differentiation. For the illustrations presented here, we only selected the original data obtained with the GM04603 iPSC line. Fig. 1A shows the experimental conditions used, according to Boissart et al (2013) [START_REF] Boissart | Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening[END_REF], to derive human iPSC with slight modifications. Figure 1B shows NPC amplification and labeling, with the selected growth and transcription factors used in this study. Figure 1C describes the map of a lentiviral vector for GFP labeling [START_REF] Vitrac | A chimeric mouse model to study human iPSCderived neurons: the case of a truncating SHANK3 mutation[END_REF].

NPC amplification and labeling

All the following steps were performed in sterile conditions under the flow hood. For their labeling, NPC were transduced with a CMV-GFP-lentivirus (green fluorescence) or with a CMV-mCherry-lentivirus (red fluorescence) as described previously [START_REF] Gouder | Three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells[END_REF]. Lentiviral vectors were prepared according to a published protocol [START_REF] Avale | Interplay of b2* nicotinic receptors and dopamine pathways in the control of spontaneous locomotion[END_REF]. The map of the CMV vector is detailed in Figure 1C. NPC were transduced with 99.5 ng of lentivirus in a 6-well culture plate filled with 500 μl of fresh culture medium for 3 hours. The volume was completed to 2 ml and incubated for the next 48 hours, and then rinsed with culture medium. For co-transplantation with two distinct populations [START_REF] Vitrac | A chimeric mouse model to study human iPSCderived neurons: the case of a truncating SHANK3 mutation[END_REF], transduced NPC should be initially purified by FACS to inject equivalent levels of green or red fluorescence intensities within cells. In the case of cotransplantation, the two populations of labeled NPC should be amplified using the same culture medium.

For amplification, NPC were thawed in T75 flasks at a density of 50,000 cells/cm 2 in 25ml of NPC medium supplemented with Rock Inhibitor Y27632 (10µM). The next day, medium was changed in order to remove the Rock inhibitor. Once confluency was reached, cells were passaged into a T175 flask. Medium was changed every 2 to 4 days during NPC amplification.

Grafting into mouse neonates

Cultured NPC were dissociated and resuspended in DPBS (10 5 cells/μl) before their transplantation into the neonatal brain. GFP-labeled cells were transplanted into the motor cortex of immunodeficient NOD/SCID mice (Charles River Laboratories), at postnatal days 1-2. Pups were first locally anesthetized using a lidocaine gel. The head of newborn pups was gently held horizontally between the operator's fingers. Bregma was used as a visible landmark to localize the injection site. We used the published coordinates Dorso-Lateral = + 1 mm, Antero-Posterior = -1 mm, depth = -1 mm to target the cortex, or depth = -2 mm to target the ventricles. Two μl of resuspended cells were slowly injected unilaterally using a Hamilton syringe (Hamilton, ref. 65460). The syringe needle was set for injecting at 1-or 2mm depth and kept in the pups' brain for 30 s following the injection before its removal. The injection lasted over 1 minute. This protocol was approved by the Institut Pasteur Ethics Committee and the French "Ministère de l'Education Nationale, de la Recherche et de l'Innovation" under reference APAFiS #20686. The accuracy of injection was monitored by immunofluorescence labeling of GFP-labeled transplants, after brain slicing at different periods post-injection. All pups per litter were injected and their survival was evaluated at ~60%. For analysis, we recommend keeping only the mice that present well-defined GFPlabeled transplants within the same cortical areas. We excluded the brains with small number of labeled transplanted cells from analysis even though they presented similar phenotypes.

In utero grafting of mouse embryos

This protocol was approved by the Institut Pasteur Ethics Committee and the French "Ministère de l'Education Nationale, de la Recherche et de l'Innovation" under reference APAFiS #25221. Swiss OF1 mice (Charles River Laboratories), were habituated to the animal facility at least 3 days and surgeries were performed at the embryonic stage of E17.5. We observed that this developmental stage allows high rate of pup's survival (~65%).

Material and equipment settings

A Hamilton syringe was connected to the pump by a plastic capillary, at the end of which a sterile glass micropipette was fixed (~ 50 µm diameter). Parafilm can be used to tightly connect plastic and glass micropipette. Before each surgery, the Hamilton syringe was filled with sterile distilled water and placed in a Harvard pump. A set of glass injection micropipettes were prepared by hand, using the needle puller, without any further autoclaving. These are the only type of needles that can be used for in utero injection. Once embryos were ready to be injected, the glass micropipette was carefully filled with cell suspension (~10 5 cells / µl), to avoid the formation of air bubbles. Injection settings were as follows: Infusion rate: 30 000 µl / min, Target volume: 2 µl, Diameter: 0.4600 mm. Depending on experimenter, the injection can be performed using a magnification loupe or under a dissection microscope, or directly without any magnification device. At the stage E17.5, the ventricles can be easily distinguished in the swiss OF1 mouse strain. Cold light and sterile surgical instruments are necessary and should be ready to use.

In utero Surgery Procedures

Before their grafting, cell suspensions were prepared according to the protocol described in section 2.2 and kept in a water bath at 37°C. For the in utero approach, the injection solution also contains 20 mM EGTA [START_REF] Linaro | Xenotransplanted human cortical neurons reveal species-specific development and functional integration into mouse visual circuits[END_REF][START_REF] Nagashima | Novel and Robust Transplantation Reveals the Acquisition of Polarized Processes by Cortical Cells Derived from Mouse and Human Pluripotent Stem Cells[END_REF] in sterile DPBS to dissociate apical junctions in the neuroepithelium and avoid cell aggregation upon transplantation. For transplantation, we injected 2 µl containing 2. 10 5 cells within one lateral ventricle of E17.5 embryos. Thirty minutes before surgery, buprenorphine (0.05 to 0.1 mg/kg) was injected subcutaneously at the back of the neck of pregnant mouse, for initial analgesia. The pregnant mouse was then anesthetized in a box with isoflurane and oxygen delivered via a continuous flow anesthesia unit and then placed on a heating pad which was also connected to the anesthesia unit. A drop of ocryl gel was applied on each eye to avoid eye dryness. The abdomen was shaved locally for ventral laparotomy at the white midline level. A betadine solution was applied on the skin, then rinsed with 70% ethanol using cotton swabs. A lidocaine gel was applied locally, before and after the laparotomy. Before starting the surgery, the sterile towel drape was positioned on the mouse abdomen. An incision of 1 to 1.5 cm was made in the lower abdomen in a manner to cut both the skin and the thin fascia layer. Abdominal organs should not be injured during this critical step. The whole uterus was carefully extracted from the abdomen, right and left ovaries were identified, and the total number of fetuses was counted. During surgery, drops of sterile PBS kept at 37°C were consistently applied on the exposed fetuses.

Then, each fetus was held to clearly identify the lateral ventricle. The fetus was stabilized between thumb and forefinger within or close to the amniotic cavity. Any fetus rotation or damage should be avoided as it can affect embryo survival. The sharp glass pipette was carefully inserted through the fetal skull, and cells were injected into each fetus, except the first one to be delivered, on each uterine horn. After the injection, the pipette was carefully withdrawn with well-controlled movements. Note that leakage of amniotic fluid should not be observed. Injections should not take more that 15-20 minutes per mouse. If the duration of injection is longer, some of the injected fetuses can be put back into the abdomen to keep them at the body temperature. Then, the uterus was carefully reentered into the abdominal cavity in a way to avoid any twist between the embryos and with a controlled holding pressure. The peritoneal cavity was rehydrated with 0.5 to 1 mL of sterile PBS to replace any fluid loss during the surgery. Then, the incision was closed with two types of sutures: for fascia and skin, we used sterile resorbable suture (Prolene TM , EH7471, Ethicon, LLC) and nonresorbable suture (Coated Vicryl, V302, Ethicon, LLC), respectively. After the surgery, the pregnant mouse was replaced in its own cage and positioned on a warming pad until the mouse was fully awake. Analgesia treatment (acetaminophen 4mg/20ml) was administered in drinking water for 3 to 5 days after the surgery, and stress (noise, excessive manipulation of cages…) was minimized to avoid preterm delivery. Hydrogel was also given to mice for one day after surgery. We observed that grafted cells' distributions and density may slightly vary between each embryo, due to a possible aggregation of cells in the pipette and during injection. We could observe that the presence of EGTA significantly reduces cell aggregation, and increase the possibility of identifying single cells within the mouse brain, as described previously [START_REF] Linaro | Xenotransplanted human cortical neurons reveal species-specific development and functional integration into mouse visual circuits[END_REF].

For a first in utero test or training, it is recommended to inject 1-2 µl of fast green or bromophenol blue solutions, without cells. If correctly injected into lateral ventricle, the solution will immediately fill the whole embryonic ventricular systems. In that case, the pregnant mouse and embryos should be sacrificed immediately by profound anesthesia followed by cervical dislocation.

Grafting into the dentate gyrus of adult mouse brain

The protocol for cell preparation is identical to that for in utero grafting. Experiments were performed with adult NOD-SCID mice (Charles River Laboratories).

As for in utero grafting, the mice received an injection of buprenorphine (0.1 mg/kg) for initial analgesia 30 min before anesthesia by isoflurane. The mouse was placed on a stereotaxic frame connected to an isoflurane unit and to a heating pad. A drop of ocryl gel was applied on each eye to avoid dryness. The skin was shaved and cleaned with betadine followed by 70% ethanol. For a local anesthesia, 150µl of Lidocaine (7 mg/kg) were injected subcutaneously at the skull level at least 10 min before the skin incision. Using a driller, two holes were done at the following coordinates A/P: -1.8; M/L: +/-1.25 for targeting the DG according to the Paxinos and Franklin Atlas [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF]. Twenty nanoliters of cell suspensions were injected bilaterally at a rate of 3nl/s in three steps along the D/V axis: 15 injections at -1.82 depth, 20 at -1.76 and 15 at -1.72 to achieve a final volume of 1 µl/ hemisphere. To ensure reproducible injection volumes, we recommend to wait 10 seconds between each injection and 5 min before removing the glass capillary from the brain tissue.

For mouse recovery, metacam (0.05mg/kg) and 200 µl of sterile saline was injected separately subcutaneously before the end of anesthesia (~50 µl/ mouse). As post-operatory care, metacam (1mg /Kg /day) diluted in diet gel was given for at least 3 days. Mice were monitored for 5 days.

Immunofluorescence labeling

For immunofluorescence labeling, mice received a lethal dose of a mixture of ketamine (Imalgen, 100 mg/kg, Merial) and Xylazine (Rompun, 10 mg/Kg, Bayer), and were transcardially perfused with 15 ml of DPBS, followed by 50 ml of ice-cold 4% paraformaldehyde (PFA). Brains were removed and post-fixed overnight. Forty µm-coronal sections were obtained using a vibratome or a microtome, collected in 12-well or 24-well culture plates containing DPBS supplemented with 0.02% azide, and stored at 4°C until use.

Free-floating sections were rinsed twice in DPBS (pH 7.4) and then incubated in DPBS solution containing 0.5% Triton X-100 and 10% serum at room temperature for 30 min to block nonspecific binding. Primary antibodies were diluted in blocking solution and incubated overnight at 4°C. In order to improve the labeling of neuron morphology, immunofluorescence was performed using an anti-GFP antibody in permeabilized conditions. An additional DAPI staining diluted in DPBS was performed for 10 min at room temperature.

Representative data

In this chapter, we describe three different approaches for grafting the human NPC which result in the integration of human cells into the brain of adult mouse. Fig. 1 illustrates the different steps of the protocol which we used for amplification, banking, and labeling of NPC before their grafting. We have previously shown that the neonatal engraftment of human NPC can lead to neurons that are able to differentiate into glutamatergic neurons [START_REF] D'alessio | Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex[END_REF][START_REF] Vitrac | A chimeric mouse model to study human iPSCderived neurons: the case of a truncating SHANK3 mutation[END_REF]. Fig. 2 illustrates the precise site of injection and the cortical graft containing a large density of neuronal cell bodies together with numerous axonal projections within distinct brain regions.

The full cartography of axonal projections in the brain has been published elsewhere [START_REF] Vitrac | A chimeric mouse model to study human iPSCderived neurons: the case of a truncating SHANK3 mutation[END_REF].

Using the same human NPC, we set up the grafting into the lateral ventricle of mouse embryo (E17.5). Our results show that two weeks after their grafting, the cells are able to integrate within the brain parenchyma and start to migrate along known migratory pathways including cingulum bundle, corpus callosum, and fimbria (Fig. 3A). At this early stage of post-grafting, a large number of cells remain close to the injection site. By contrast, the cells are found to be more dispersed after one month and in the process of integrating the distinct brain regions including the striatum (Fig. 3A,B). Two months post-grafting, the cells start to differentiate into excitatory cells (Fig. 3C). At this stage, the co-localization of VGlut labeling with the human cells is observed both in neuronal cell bodies and projections (Fig. 3C). Fig. 4 illustrates the grafting of the human NPC into the dentate gyrus (DG) of adult immunodeficient mice, three weeks post-grafting. Our data show that cells can survive and integrate within the hippocampal structures following a stereotaxic injection in adult mice.

Notes

The three humanized mouse models

The use of humanized chimeric mouse models allows the study of neuronal development and associated pathogenesis and neurodegenerative diseases using human cells in an in vivo condition. Under the experimental approaches described here, the neonatal grafting allows the grafted progenitors to predominantly differentiate into excitatory neurons which can mature, migrate, and project their axons in different brain regions. The iPSC technology has offered the possibility to study some specific phenotypes, including cell differentiation and maturation, neuronal morphology and functional activities by electrophysiological recordings of cultured cells under in vitro conditions [START_REF] Espuny-Camacho | Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo[END_REF]. However, in vitro models using human iPSC-derived neurons are limited as they do not offer the possibility to study disease pathogenesis at different stages of brain maturation within a such complex environment, and in the presence of external cues that are necessary for neuronal patterning. As shown in which has allowed the discovery of a panel of morphogenetic abnormalities that are linked to early development of neurons with ASD-associated mutations [START_REF] Vitrac | A chimeric mouse model to study human iPSCderived neurons: the case of a truncating SHANK3 mutation[END_REF][START_REF] Kathuria | Stem cell-derived neurons from autistic individuals with SHANK3 mutation show morphogenetic abnormalities during early development[END_REF]. It should also be noted that transplanted human PSC-derived NPC can be tumorigenic, if contaminated with residual pluripotent stem cells with sustained proliferation of residual stem cells [START_REF] Chung | Genetic selection of sox1GFP-expressing neural precursors removes residual tumorigenic pluripotent stem cells and attenuates tumor formation after transplantation[END_REF]. As shown in Fig. 1, we used the SMAD inhibitor SB431542, based on previously reported studies [START_REF] Boissart | Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening[END_REF], in order to avoid abnormal cell proliferation. For in utero grafting, we observed large densities of graft-derived fibers projecting into migratory pathways and in the host tissue as early as 15 days post-grafting (Fig. 3). The in utero grafting method presents some advantages as it can be applied to immunocompetent mice with no graft rejection and no inflammation at injection sites (data not shown), while mouse neurogenesis continues which may favor further development of human cells.

Our results indicate that the cells can survive at least for 3 months after grafting and give rise to glutamatergic neurons (Fig. 3). We also observed that they develop dendritic spines after neonatal grafting (data not shown), using the same GFP-labeled human iPSC line [START_REF] Vitrac | A chimeric mouse model to study human iPSCderived neurons: the case of a truncating SHANK3 mutation[END_REF]. To our knowledge, there is no report on the in utero grafting of human iPSC-derived neuronal cells in rodent brain. The available data from using mouse embryonic cells grafted in the brain of rat embryos, were analyzed within 7 weeks post-grafting [START_REF] Brüstle | Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats[END_REF]. However, human cells are expected to be found uniformly throughout the brain regions, including hippocampus, cerebral cortex and striatum at later stages, up to 13 months, as observed in neonatally grafted mice [START_REF] Chen | Humanized neuronal chimeric mouse brain generated by neonatally engrafted human iPSC-derived primitive neural progenitor cells[END_REF]. Finally, we describe the grafting of NPC within hippocampus of the adult mice. This grafting method may help targeting a specific brain region, and to analyze more specifically how grafted cells can integrate within specific circuits. We illustrate this approach with NPC giving rise to excitatory neurons after grafting into the dentate gyrus of hippocampus. The same approach could be applied to interneurons that can be derived from human iPSC as well [START_REF] Ahn | Differentiation of human pluripotent stem cells into Medial Ganglionic Eminence vs. Caudal Ganglionic Eminence cells[END_REF]. Interestingly, interneurons represent a large and diverse population of hippocampal neurons that are critical for information and memory processing. By grafting well-characterized mature interneurons, it is possible to identify more specifically how a specific human interneuron subtype may morphologically and functionally interact within the surrounding host cells.

Applications for neurodevelopmental disorders

Neurodevelopmental disorders are either due to various inherited mutations or acquired causal factors, and lead to dysfunction of brain development and cognitive deficits.

The pathogenesis of neurodevelopmental disorders has not been well elucidated and no effective treatment has so far been found for these disorders. The establishment of accurate models which allow to investigate neurological disorders represents an important step towards better understanding and appropriate treatment of neurodevelopmental pathologies. To study the brain-associated diseases in vitro has been not possible, and to obtain the tissues from human brain represents a difficult alternative. The use of patient-derived iPSC however, offers a unique opportunity to investigate pathological changes that may occur in the brain at early stages of neuronal development. In addition, human iPSC provide an auto-renewal source of cells which are identical to the donor [START_REF] Nestor | Human Inducible Pluripotent Stem Cells and Autism Spectrum Disorder: Emerging Technologies[END_REF]. Under the action of specific growth and transcription factors, they can differentiate into neurons of different phenotypes that are found within the six cortical layers, including excitatory and inhibitory neurons [START_REF] Van Den Ameele | Thinking out of the dish: what to learn about cortical development using pluripotent stem cells[END_REF]. The grafting of iPSC-derived neurons into mouse brain offers the possibility to decipher cellular mechanisms in different temporal windows, from the neurogenic stages with undifferentiated cells to later stages of well-differentiated mature neurons, under control and pathological conditions. For genetic diseases, the iPSC from patients carry the genetic pattern of mutations from these donors and should display similar pathological and phenotypical changes. gene is also associated with pathogenesis and neuropathology of ASD, which involves synaptic dysfunctions and an imbalance in synaptic excitation/inhibition ratio. Using iPSC-derived neurons from ASD patients, we observed altered spinogenesis in all the patients who were included in the study [START_REF] Gouder L | Altered spinogenesis in iPSC-derived cortical neurons from patients with autism carrying de novo SHANK3 mutations[END_REF]. In a subsequent study, we used the same cells from a control individual at precursor stages (NPC), and transplanted them into the cortex of newborn mouse brain [START_REF] Vitrac | A chimeric mouse model to study human iPSCderived neurons: the case of a truncating SHANK3 mutation[END_REF]. We analyzed the integration of NPC, the maturation of the derived excitatory neurons and their axonal projections into the adult brain. We then co-transplanted the NPC from a control individual and from a patient carrying one de novo heterozygous mutation in SHANK3. In human neurons with the SHANK3 mutation, we observed a reduction in cell soma size of selective neuronal populations and in axonal projections, as compared to control neurons, at 30 days post-transplantation. However, we did not observe any alteration in spinogenesis at this early stage, which are expected to occur at later stages of development.

This indicates that in vivo models can recapitulate the early stages of the disease, which may help understand the initial mechanisms before the appearance of the well-described phenotypes observed in mouse models or in human cells kept in culture.

Conclusion

One of the strong aspects of our different grafting approaches is that humanized mouse models can be used for both neurodevelopmental as well as neurodegenerative diseases, depending on the stage at which the phenotypes of grafted cells are evaluated. At the early stages of development, regulation of cortical neurogenesis is very important as it is involved in neuronal fate [START_REF] Villalba | The regulation of cortical neurogenesis[END_REF]. For example, recent discoveries point out the permissive role of the amyloid precursor protein (APP) in the regulation of human cortical neurogenesis [START_REF] Shabani | The Amyloid Precursor Protein regulates human cortical neurogenesis[END_REF]. APP is also the precursor of b-amyloid peptide which can lead to progressive accumulation of bamyloid oligomers, followed by their aggregation in amyloid plaques, one of the major hallmarks of the neurodegenerative Alzheimer's disease (AD). It would be possible to test whether the physiological functions of APP are related to the onset of AD, using in vivo approaches such as mouse chimeric models, xenografted in utero and appropriate genetic tools, to follow and control the production of endogenous APP within the whole brain or locally in specific brain regions. Humanized mouse models not only offer the possibilities to decipher the early events which underlie neuronal maturation and plasticity but also to investigate the pathological events that occur in the brain of patients with neurological disorders, including those with AD. Such models should also facilitate the preclinical evaluation of therapeutic measures and treatments as well as their consequences within the human neurons, in vivo. Finally, these models can help to set up appropriate transplantation therapies using neural stem cells. The xenografting strategies offer the possibility to compensate neural loss and neural tissue injury and should allow the discovery of therapeutic strategies for the future, in the context of both developmental disorders and degenerative diseases. One main challenge will be to face the complexity of human brain and consider its unique spatial and temporal development.

Figure 1 .-

 1 Figure 1.

  For autism spectrum disorders (ASD), recent studies have shown that the events underlying such disorders, can occur during neonatal development, together with strong synaptic defects that are known to occur at later stages of development. In a previous in vitro study, we investigated the iPSC-derived pyramidal cortical neurons from patients with ASD carrying de novo heterozygous mutations in the postsynaptic SHANK3 protein. SHANK3 is a synaptic scaffolding protein located at the postsynaptic density of excitatory synapses [54]. The haploinsufficiency of SHANK3 gene provokes a developmental disorder known as the 22q13.3 deletion syndrome (or Phelan-McDermid syndrome). SHANK3

Legend to Figure 1 :Legend to Figure 2 :

 12 In vitro cell differentiation, amplification and labeling before engraftment. A. Human iPSC are first amplified and then differentiated in early cortical progenitors, then to NPC with further amplification before banking. B. Description of NPC amplification and labeling before grafting into mouse brain. GFP labeling can be started directly from the stock of frozen NPC. C. Schematic representation of the lentiviral construct for transduction of NPC with GFP (green fluorescence) using a human cytomegalovirus promoter (CMVp). Engrafting of human iPSC-derived NPC into the cortex of newborn mice. A. Intracranial injection of human iPSC-derived NPC into the cortex of neonatal mice. The Lambda and Bregma landmarks are used to target the motor cortex as indicated in red. The injection site is located approximately 1 mm posterior to the bregma and 1 mm lateral to the sagittal suture, with 1 mm depth, as indicated by the red symbol. B: Representative engraftments into the cortex of 50-day old mice after neonatal injection of NPC. Graft location within the somatosensory cortex. Most of GFP-labeled neurons remain within the graft site, and extend their dendrites. C. Axonal projections within the somato-sensory cortex (Bregma ≈ -0.955 mm). D. Axonal projections in the somato-sensory cortex (Bregma ≈ 1.545 mm). E. Long-range axonal projections in the retrohippocampal region (Bregma ≈ -2.78 mm) F. Long-range axonal projections in the somato-sensory cortex (Bregma ≈ -2.78 mm). Scale bars = 200 µm.

  Abbreviations. AP: Action potentials; ASD: autism spectrum disorders; CGE: Caudal ganglionic

Figure 1 BDNF: 20

 120 Figure 1

Figure 2

 2 Figure 2

Table 1 ,

 1 For neonatal grafting, it has been shown that grafted NPC appear to respond to region specific signals and are also responsive to neural patterning cues in vitro[4; 12; 41]. These human cells progressively expand and migrate in the murine forebrain. It has been difficult to understand to what extent the undifferentiated NPC or the immature neurons can integrate and recapitulate physiological neurogenesis in vivo. Moreover, most studies have been limited due to the use of immunodeficient NOD-SCID mice, which have the tendency to form tumors at 7-8 months of age[START_REF] Santagonisto | Pathology of aging in NOD scid gamma female mice[END_REF]. So far, these issues have not been considered for studying neurodevelopmental disorders. Interest has been focused on early stages of neuronal maturation,

the neonatal grafting of iPSC-derived neuronal progenitors has been successfully used by several authors. By comparing neonatal and in utero grafting, we observed that in both cases, the engrafted cells form initial clusters during the first month post-grafting which tend to remain over time, at least up to 7 months

[START_REF] D'alessio | Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex[END_REF]

.

Table 1 : Reported studies using xenotransplantation of human stem cell-derived neurons

 1

	Human cell	Host	Brain region	Graft Integration	Functionality Ref.
	origin
	Fetal brain	Rat embryo	Brain ventricles	Widespread	n.d.	[22]
	cells			incorporation of neurons
				Migration along the routes
	Embryonic	Adult rat	SVZ & Hp	taken by endogenous
	NSC			precursors. Substantial	n.d.	[23]
				migration within the
				striatum
	Fetal brain	Mouse	Brain ventricles	Proliferation, migration,
	cells	neonate		and neural differentiation	n.d.	[24]
				(7MPT)
				At 16-17 weeks of
		Monkey		gestation:
	NSC	fetus at 12-	Brain ventricles	Two subpopulations:	n.d.	[25]
		13 week-		-Differentiated neurons &
		gestation		glia; -Undifferentiated
				cells (SVZ)
				At 1-4 WPT:
				Incorporation of cells in
				cortex, Hp, OB, septum,
	ESC-derived NPC	Mouse neonate	Brain ventricles	thalamus, hypothalamus, striatum midbrain and	n.d.	[26]
				some white-matter regions
				-Differentiation into
				neurons and astrocytes
			In ovo grafting	6-7 days post-surgery
	ESC	Chick	into somites;	Differentiation and	n.d.	[27]
		embryo	1.5-2 days of	migration into DRG,
			development	neural tube
				Differentiation into	Generation
	Immortalized	Rat with		neurons & astrocytes in	of AP &	[28]
	NS-like cells	focal	i.v. injection	lesioned areas; survival up	synaptic
		cerebral		to 56 days after grafting	integration-
		ischemia		(cortex & striatum)	Miniature
					EPSC
		Mouse		2 MPT:	8 MPT:
	Differentiated	embryo	Brain ventricles	Widespread incorporation	Action	[29]
	ESC	(E14)		including within cortex	Potentials
				and hippocampus
				Integration into SVZ;
		Young /		migration along the rostral
	ESC-derived	adult rat	Brain ventricles	migratory stream to	n.d.	[30]
	NPC	brain		olfactory bulb;
				contribution to
				neurogenesis
	ESC-derived	6-OHDA-
	DA neurons (+ astrocytes)	lesioned adult rat	Striatum	recovery Substantial neuronal	testing Behavioral	[31]
	ESC-derived			Xenograft overgrowth
	striatal	Adult rat	Striatum	& persistent proliferation	n.d.	[32]
	progenitors			of NPC
		Parkinsonian		4-5 MPT. No neural	Membrane
	ESC-derived	models in	Striatum	overgrowth. In vivo	potential	[33]
	DA neurons	mouse, rat &		survival. Restoration of	oscillations
		monkey		behavioral symptoms

Acknowledgements

The authors are thankful to Dr. Alexandra Benchoua who performed the initial iPSC reprogramming. The "Fondation Recherche Alzheimer" provides the funding for CT's PhD. The Pasteur-Paris University International doctoral program (PPU) provides the funding for MLP's PhD. French Ministry of Education and the LabEx BioPsy provided the funding for AV's PhD. Other funding for this study were provided by grants from the French National Research Agency ANR (ANR-17-NEU3-0004; IPS BRAIN), from ERA-NET NEURON (iPSC & Brain), and from the "France Alzheimer" foundation. We gratefully acknowledge the UtechS Photonic Bioimaging (Imagopole), C2RT, Institut Pasteur, supported by the French National Research Agency (France BioImaging; ANR-10-INSB-04; Investments for the future).

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