High and low permeability of human pluripotent stem cell-derived blood-brain barrier models depend on epithelial or endothelial features.

The search for reliable human blood-brain barrier (BBB) models represents a challenge for the development/testing of strategies aiming to enhance brain delivery of drugs. Human-induced pluripotent stem cells (hiPSCs) have raised hopes in the development of predictive BBB models. Differentiating strategies are thus required to generate endothelial cells (ECs), a major component of the BBB. Several hiPSC-based protocols have reported the generation of in vitro models with significant differences in barrier properties. We studied in depth the properties of iPSCs byproducts from two protocols that have been established to yield these in vitro barrier models. Our analysis/study reveals that iPSCs derivatives endowed with EC features yield high permeability models while the cells that exhibit outstanding barrier properties show principally epithelial cell-like (EpC) features. We found that models containing EpC-like cells express tight junction proteins, transporters/efflux pumps and display a high functional tightness with very low permeability, which are features commonly shared between BBB and epithelial barriers. Our study demonstrates that hiPSC-based BBB models need extensive characterization beforehand and that a reliable human BBB model containing EC-like cells and displaying low permeability is still needed.

The Helico Maze Detects Early Impairment of Reference Memory at Three Months of Age in the 5XFAD Mouse Model of Alzheimer's Disease.

BACKGROUND: The 5XFAD model of Alzheimer's disease (AD) bearing five familial mutations of Alzheimer's disease on human APP and PSEN1 transgenes shows deposits of amyloid-ß peptide (Aß) as early as 2 months, while deficits in long-term memory can be detected at 4 months using the highly sensitive olfactory-dependent tests that we previously reported. OBJECTIVE: Given that detecting early dysfunctions in AD prior to overt pathology is of major interest in the field, we sought to detect memory deficits at earlier stages of the disease in 3-month-old male 5XFAD mice. METHODS: To this end, we used the Helico Maze, a behavioral task that was recently developed and patented. This device allows deeper analysis of learning and subcategories of hippocampal-dependent long-term memory using olfactory cues. RESULTS: Eight male 5XFAD and 6 male wild-type (WT: C57Bl6 background) mice of 3 months of age were tested in the Helico Maze. The results demonstrated, for the first time, a starting deficit of pure reference long-term memory. Interestingly, memory impairment was clearly correlated with Aß deposits in the hippocampus. While we also found significant differences in astrogliosis between 5XFAD and WT mice, this was not correlated with memory abilities. CONCLUSION: Our results underline the efficiency of this new olfactory-dependent behavioral task, which is easy to use, with a small cohort of mice. Using the Helico Maze may open new avenues to validate the efficacy of treatments that target early events related to the amyloid-dependent pathway of the disease and AD progression.

Syngeneic Transplantation of Olfactory Ectomesenchymal Stem Cells Restores Learning and Memory Abilities in a Rat Model of Global Cerebral Ischemia.

Stem cells are considered as promising tools to repair diverse tissue injuries. Among the different stem cell types, the "olfactory ectomesenchymal stem cells" (OE-MSCs) located in the adult olfactory mucosa stand as one of the best candidates. Here, we evaluated if OE-MSC grafts could decrease memory impairments due to ischemic injury. OE-MSCs were collected from syngeneic F344 rats. After a two-step global cerebral ischemia, inducing hippocampal lesions, learning abilities were evaluated using an olfactory associative discrimination task. Cells were grafted into the hippocampus 5 weeks after injury and animal's learning abilities reassessed. Rats were then sacrificed and the brains collected for immunohistochemical analyses. We observed significant impairments in learning and memory abilities following ischemia. However, 4 weeks after OE-MSC grafts, animals displayed learning and memory performances similar to those of controls, while sham rats did not improve them. Immunohistochemical analyses revealed that grafts promoted neuroblast and glial cell proliferation, which could permit to restore cognitive functions. These results demonstrated, for the first time, that syngeneic transplantations of OE-MSCs in rats can restore cognitive abilities impaired after brain injuries and provide support for the development of clinical studies based on grafts of OE-MSCs in amnesic patients following brain injuries.

Isolation and characterization of olfactory ecto-mesenchymal stem cells from eight mammalian genera.

BACKGROUND: Stem cell-based therapies are an attractive option to promote regeneration and repair defective tissues and organs. Thanks to their multipotency, high proliferation rate and the lack of major ethical limitations, "olfactory ecto-mesenchymal stem cells" (OE-MSCs) have been described as a promising candidate to treat a variety of damaged tissues. Easily accessible in the nasal cavity of most mammals, these cells are highly suitable for autologous cell-based therapies and do not face issues associated with other stem cells. However, their clinical use in humans and animals is limited due to a lack of preclinical studies on autologous transplantation and because no well-established methods currently exist to cultivate these cells. Here we evaluated the feasibility of collecting, purifying and amplifying OE-MSCs from different mammalian genera with the goal of promoting their interest in veterinary regenerative medicine. Biopsies of olfactory mucosa from eight mammalian genera (mouse, rat, rabbit, sheep, dog, horse, gray mouse lemur and macaque) were collected, using techniques derived from those previously used in humans and rats. The possibility of amplifying these cells and their stemness features and differentiation capability were then evaluated. RESULTS: Biopsies were successfully performed on olfactory mucosa without requiring the sacrifice of the donor animal, except mice. Cell populations were rapidly generated from olfactory mucosa explants. These cells displayed similar key features of their human counterparts: a fibroblastic morphology, a robust expression of nestin, an ability to form spheres and similar expression of surface markers (CD44, CD73). Moreover, most of them also exhibited high proliferation rates and clonogenicity with genus-specific properties. Finally, OE-MSCs also showed the ability to differentiate into mesodermal lineages. CONCLUSIONS: This article describes for the first time how millions of OE-MSCs can be quickly and easily obtained from different mammalian genera through protocols that are well-suited for autologous transplantations. Moreover, their multipotency makes them relevant to evaluate therapeutic application in a wide variety of tissue injury models. This study paves the way for the development of new fundamental and clinical studies based on OE-MSCs transplantation and suggests their interest in veterinary medicine.

Grafts of Olfactory Stem Cells Restore Breathing and Motor Functions after Rat Spinal Cord Injury.

The transplantation of olfactory ecto-mesenchymal stem cells (OEMSCs) could be a helpful therapeutic strategy for spinal cord repair. Using an acute rat model of high cervical contusion that provokes a persistent hemidiaphragmatic and foreleg paralysis, we evaluated the therapeutic effect of a delayed syngeneic transplantation (two days post-contusion) of OEMSCs within the injured spinal cord. Respiratory function was assessed using diaphragmatic electromyography and neuroelectrophysiological recordings of phrenic nerves (innervating the diaphragm). Locomotor function was evaluated using the ladder-walking locomotor test. Cellular reorganization in the injured area was also studied using immunohistochemical and microscopic techniques. We report a substantial improvement in breathing movements, in activities of the ipsilateral phrenic nerve and ipsilateral diaphragm, and also in locomotor abilities four months post-transplantation with nasal OEMSCs. Moreover, in the grafted spinal cord, axonal disorganization and inflammation were reduced. Some grafted stem cells adopted a neuronal phenotype, and axonal sparing was observed in the injury site. The therapeutic effect on the supraspinal command is presumably because of both neuronal replacements and beneficial paracrine effects on the injury area. Our study provides evidence that nasal OEMSCs could be a first step in clinical application, particularly in patients with reduced breathing/locomotor movements.

Chronic treatments with a 5-HT4 receptor agonist decrease amyloid pathology in the entorhinal cortex and learning and memory deficits in the 5xFAD mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the main cause of dementia and a major health issue worldwide. The complexity of the pathology continues to challenge its comprehension and the implementation of effective treatments. In the last decade, a number of possible targets of intervention have been pointed out, among which the stimulation of 5-HT4 receptors (5-HT4Rs) seems very promising. 5-HT4R agonists exert pro-cognitive effects, inhibit amyloid-ß peptide (Aß) production and therefore directly and positively impact AD progression. In the present work, we investigated the effects of RS 67333, a partial 5-HT4R agonist, after chronic administration in the 5xFAD mouse model of AD. 5xFAD male mice and their wild type (WT) male littermates received either RS 67333 or vehicle solution i.p., twice a week, for 2 or 4 months. Cognitive performance was evaluated in a hippocampal-dependent behavioral task, the olfactory tubing maze (OTM). Mice were then sacrificed to evaluate the metabolism of the amyloid precursor protein (APP), amyloidosis and neuroinflammatory processes. No beneficial effects of RS 67333 were observed in 5xFAD mice after 2 months of treatment, while 5xFAD mice treated for 4 months showed better cognitive abilities compared to vehicle-treated 5xFAD mice. The beneficial effects of RS 67333 on learning and memory correlated with the decrease in both amyloid plaque load and neuroinflammation, more specifically in the entorhinal cortex. The most significant improvements in learning and memory and reduction of pathology stigmata were observed after the 4-month administration of RS 67333, demonstrating that treatment duration is important to alleviate amyloidosis and glial reactivity, particularly in the entorhinal cortex. These results confirm the 5-HT4R as a promising target for AD pathogenesis and highlight the need for further investigations to characterize fully the underlying mechanisms of action.

From Blood to Lesioned Brain: An In Vitro Study on Migration Mechanisms of Human Nasal Olfactory Stem Cells.

Stem cell-based therapies critically rely on selective cell migration toward pathological or injured areas. We previously demonstrated that human olfactory ectomesenchymal stem cells (OE-MSCs), derived from an adult olfactory lamina propria, migrate specifically toward an injured mouse hippocampus after transplantation in the cerebrospinal fluid and promote functional recoveries. However, the mechanisms controlling their recruitment and homing remain elusive. Using an in vitro model of blood-brain barrier (BBB) and secretome analysis, we observed that OE-MSCs produce numerous proteins allowing them to cross the endothelial wall. Then, pan-genomic DNA microarrays identified signaling molecules that lesioned mouse hippocampus overexpressed. Among the most upregulated cytokines, both recombinant SPP1/osteopontin and CCL2/MCP-1 stimulate OE-MSC migration whereas only CCL2 exerts a chemotactic effect. Additionally, OE-MSCs express SPP1 receptors but not the CCL2 cognate receptor, suggesting a CCR2-independent pathway through other CCR receptors. These results confirm that OE-MSCs can be attracted by chemotactic cytokines overexpressed in inflamed areas and demonstrate that CCL2 is an important factor that could promote OE-MSC engraftment, suggesting improvement for future clinical trials.

Global cerebral ischemia in rats leads to amnesia due to selective neuronal death followed by astroglial scar formation in the CA1 layer.

Global Cerebral Ischemia (GCI) occurs following cardiac arrest or neonatal asphyxia and leads to harmful neurological consequences. In most cases, patients who survive cardiac arrest develop severe cognitive and motor impairments. This study focused on learning and memory deficits associated with brain neuroanatomical reorganization that appears after GCI. The four-vessel occlusion (4VO) model was performed to produce a transient GCI. Hippocampal lesions in ischemic rats were visualized using anatomical Magnetic Resonance Imaging (aMRI). Then, the learning and memory abilities of control and ischemic (bilaterally or unilaterally) rats were assessed through the olfactory associated learning task. Finally, a "longitudinal" histological study was carried out to highlight the cellular reorganizations occurring after GCI. We demonstrated that the imaging, behavioral and histological results are closely related. In fact, aMRI revealed the appearance of hyper-intense signals in the dorsal hippocampus at day 3 post-GCI. Consequently, we showed a rise in cell proliferation (Ki 67+ cells) and endogenous neurogenesis especially in the dentate gyrus (DG) at day 3 post-GCI. Then, hyper-intense signals in the dorsal hippocampus were confirmed by strong neuronal losses in the CA1 layer at day 7 post-GCI. These results were linked with severe learning and memory impairments only in bilaterally ischemic rats at day 14 post-GCI. This amnesia was accompanied by huge astroglial and microglial hyperactivity at day 30 post-GCI. Finally, Nestin+ cells and astrocytes gave rise to astroglial scars, which persisted 60days post-GCI. In the light of these results, the 4VO model appears a reliable method to produce amnesia in order to study and develop new therapeutic strategies.

MT5-MMP Promotes Alzheimer's Pathogenesis in the Frontal Cortex of 5xFAD Mice and APP Trafficking in vitro.

We previously reported that deficiency of membrane-type five matrix metalloproteinase (MT5-MMP) prevents amyloid pathology in the cortex and hippocampus of 5xFAD mice, and ameliorates the functional outcome. We have now investigated whether the integrity of another important area affected in Alzheimer's disease (AD), the frontal cortex, was also preserved upon MT5-MMP deficiency in 4-month old mice at prodromal stages of the pathology. We used the olfactory H-maze (OHM) to show that learning impairment associated with dysfunctions of the frontal cortex in 5xFAD was prevented in bigenic 5xFAD/MT5-MMP-/- mice. The latter exhibited concomitant drastic reductions of amyloid beta peptide (Aß) assemblies (soluble, oligomeric and fibrillary) and its immediate precursor, C99. Simultaneously, astrocyte reactivity and tumor necrosis factor alpha (TNF-α) levels were also lowered. Moreover, MT5-MMP deficiency induced a decrease in N-terminal soluble fragments of amyloid precursor protein (APP), including soluble APPα (sAPPα), sAPPß and the MT5-MMP-linked fragment of 95 kDa, sAPP95. However, the lack of MT5-MMP did not affect the activity of ß- and γ-secretases. In cultured HEKswe cells, transiently expressed MT5-MMP localized to early endosomes and increased the content of APP and Aß40 in these organelles, as well as Aß levels in cell supernatants. This is the first evidence that the pro-amyloidogenic features of MT5-MMP lie, at least in part, on the ability of the proteinase to promote trafficking into one of the amyloidogenic subcellular loci. Together, our data further support the pathogenic role of MT5-MMP in AD and that its inhibition improves the functional and pathological outcomes, in this case in the frontal cortex. These data also support the idea that MT5-MMP could become a novel therapeutic target in AD.

The FVB/N mice: A well suited strain to study learning and memory processes using olfactory cues.

The FVB/N mice are well suited to generate transgenic animals. These mice are also particularly sensitive to seizures and neurodegeneration induced by systemic administration of chemoconvulsants and are very useful to model epilepsy. However, previous studies report strong cognitive and visual impairments suggesting this background unsuitable for behavioral analysis. In this study, we assessed and compared learning abilities of FVB/N mice to the well characterized C57BL/6 strain using the olfactory tubing maze, a non-visual hippocampus-dependent task in which the mice were trained to learn odor-reward associations. Exploratory behavior and spontaneous locomotor activity were then compared using the open field test. We demonstrated that FVB/N mice were able to learn the task, reaching at the end of the test a high percentage of correct responses. Interestingly, the performance of the FVB/N mice was at least similar to that of the C57BL/6 mice. Moreover, in contrast to previous reports, the FVB/N mice displayed a spontaneous locomotor activity lower than C57BL/6 mice. Our study demonstrated that FVB/N mice are not cognitively impaired and that their learning and memory performance can be assessed when the task is based on olfaction rather than vision.

A unique method for the isolation of nasal olfactory stem cells in living rats.

Stem cells are attractive tools to develop new therapeutic strategies for a variety of disorders. While ethical and technical issues, associated with embryonic, fetal and neural stem cells, limit the translation to clinical applications, the nasal stem cells identified in the human olfactory mucosa stand as a promising candidate for stem cell-based therapies. Located in the back of the nose, this multipotent stem cell type is readily accessible in humans, a feature that makes these cells highly suitable for the development of autologous cell-based therapies. However, preclinical studies based on autologous transplantation of rodent olfactory stem cells are impeded because of the narrow opening of the nasal cavity. In this study, we report the development of a unique method permitting to quickly and safely biopsy olfactory mucosa in rats. Using this newly developed technique, rat stem cells expressing the stem cell marker Nestin were successfully isolated without requiring the sacrifice of the donor animal. As an evidence of the self-renewal capacity of the isolated cells, several millions of rat cells were amplified from a single biopsy within four weeks. Using an olfactory discrimination test, we additionally showed that this novel biopsy method does not affect the sense of smell and the learning and memory abilities of the operated animals. This study describes for the first time a methodology allowing the derivation of rat nasal cells in a way that is suitable for studying the effects of autologous transplantation of any cell type present in the olfactory mucosa in a wide variety of rat models.

Onset of hippocampus-dependent memory impairments in 5XFAD transgenic mouse model of Alzheimer's disease.

The 5XFAD mice are an early-onset transgenic model of Alzheimer's disease (AD) in which amyloid plaques are first observed between two and four months of age in the cortical layer five and in the subiculum of the hippocampal formation. Although cognitive alterations have been described in these mice, there are no studies that focused on the onset of hippocampus-dependent memory deficits, which are a hallmark of the prodromal stage of AD. To identify when the first learning and memory impairments appear, 5XFAD mice of two, four, and six months of age were compared with their respective wild-type littermates using the olfactory tubing maze, which is a very sensitive hippocampal-dependent task. Deficits in learning and memory started at four months with a substantial increase at six months of age while no olfactory impairments were observed. The volumetric study using magnetic resonance imaging of the whole brain and specific areas (olfactory bulb, striatum, and hippocampus) did not reveal neuro-anatomical difference. Slight memory deficits appeared at 4 months of age in correlation with an increased astrogliosis and amyloid plaque formation. This early impairment in learning and memory related to the hippocampal dysfunction is particularly suited to assess preclinical therapeutic strategies aiming to delay or suppress the onset of AD.

Isolation of adult stem cells from the human olfactory mucosa.

The olfactory mucosa, located in the nasal cavity, is the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. In addition, this organ is home of a continuing neurogenesis that is sustained by a large population of stem cells. Here, we describe a method for biopsying olfactory mucosa from human nasal cavities and isolating multipotent adult stem cells that can be used to either identify biomarkers in brain disorders or repair the pathological/traumatized nervous system.

Role of matrix metalloproteinases in migration and neurotrophic properties of nasal olfactory stem and ensheathing cells.

Adult olfactory ectomesenchymal stem cells (OE-MSCs) and olfactory ensheathing cells (OECs), both from the nasal olfactory lamina propria, display robust regenerative properties when transplanted into the nervous system, but the mechanisms supporting such therapeutic effects remain unknown. Matrix metalloproteinases (MMPs) are an important family of proteinases contributing to cell motility and axonal outgrowth across the extracellular matrix (ECM) in physiological and pathological conditions. In this study, we have characterized for the first time in nasal human OE-MSCs the expression profile of some MMPs currently associated with cell migration and invasiveness. We demonstrate different patterns of expression for MMP-1, MMP-2, MMP-9, and MT1-MMP upon cell migration when compared with nonmigrating cells. Our results establish a correspondence between the localization of these proteinases in the migration front with the ability of cells to migrate. Using various modulators of MMP activity, we also show that at least MMP-2, MMP-9, and MT1-MMP contribute to OE-MSC migration in an in vitro 3D test. Furthermore, we demonstrate under the same conditions of culture used for in vivo transplantation that OE-MSCs and OECs secrete neurotrophic factors that promote neurite outgrowth of cortical and dorsal root ganglia (DRG) neurons, as well as axo-dendritic differentiation of cortical neurons. These effects were abolished by the depletion of MMP-2 and MMP-9 from the culture conditioned media. Altogether, our results provide the first evidence that MMPs may contribute to the therapeutic features of OE-MSCs and OECs through the control of their motility and/or their neurotrophic properties. Our data provide new insight into the mechanisms of neuroregeneration and will contribute to optimization of cell therapy strategies.

Evidence for early cognitive impairment related to frontal cortex in the 5XFAD mouse model of Alzheimer's disease.

The frontal cortex is a brain structure that plays an important role in cognition and is known to be affected in Alzheimer's disease (AD) in humans. Over the past years, transgenic mouse models have been generated to recapitulate the main features of this disease, including cognitive impairments. This study investigates frontal cortex dependent learning abilities in one of the most early-onset transgenic murine model of AD, the 5XFAD mice. We compared frontal performance of 2-, 4-, and 6-month-old 5XFAD mice with their wild-type littermates using a newly developed automated device, the olfactory H-maze, in which mice have to discover three different rules consecutively according to the delayed reaction paradigm. We report early cognitive deficits related to frontal cortex appearing in 4-month-old 5XFAD mice before hippocampal-dependent learning and memory impairment, in relation with neuropathologic processes such as strong gliosis and emerging amyloid plaques. The present results demonstrate that the olfactory H-maze is a very sensitive and simple experimental paradigm that allows assessment of frontal functions in transgenic mice and should be useful to test pre-clinical therapeutic approaches to alter the course of AD.

Isolating nasal olfactory stem cells from rodents or humans.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell(1). Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)(2-4) or neurodegenerative (e.g. Parkinson, Alzheimer) diseases(4,5). Olfactory mucosa can be used for either comparative molecular studies(4,6) or in vitro experiments on neurogenesis(3,7). The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria(8-10). We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)(11). Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease(4). We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma(12,13), a cochlear damage(14) or in an animal models of Parkinson's disease(15) or amnesia(16). In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

Engraftment of human nasal olfactory stem cells restores neuroplasticity in mice with hippocampal lesions.

Stem cell-based therapy has been proposed as a potential means of treatment for a variety of brain disorders. Because ethical and technical issues have so far limited the clinical translation of research using embryonic/fetal cells and neural tissue, respectively, the search for alternative sources of therapeutic stem cells remains ongoing. Here, we report that upon transplantation into mice with chemically induced hippocampal lesions, human olfactory ecto-mesenchymal stem cells (OE-MSCs) - adult stem cells from human nasal olfactory lamina propria - migrated toward the sites of neural damage, where they differentiated into neurons. Additionally, transplanted OE-MSCs stimulated endogenous neurogenesis, restored synaptic transmission, and enhanced long-term potentiation. Mice that received transplanted OE-MSCs exhibited restoration of learning and memory on behavioral tests compared with lesioned, nontransplanted control mice. Similar results were obtained when OE-MSCs were injected into the cerebrospinal fluid. These data show that OE-MSCs can induce neurogenesis and contribute to restoration of hippocampal neuronal networks via trophic actions. They provide evidence that human olfactory tissue is a conceivable source of nervous system replacement cells. This stem cell subtype may be useful for a broad range of stem cell-related studies.