CXCL10 is a crucial chemoattractant for efficient intranasal delivery of mesenchymal stem cells to the neonatal hypoxic-ischemic brain.

BACKGROUND: Hypoxic-Ischemic Encephalopathy (HIE) is a leading cause of mortality and morbidity in newborns. Recent research has shown promise in using intranasal mesenchymal stem cell (MSC) therapy if administered within 10 days after Hypoxia-Ischemia (HI) in neonatal mice. MSCs migrate from the nasal cavity to the cerebral lesion in response to chemotactic cues. Which exact chemokines are crucial for MSC guidance to the HI lesion is currently not fully understood. This study investigates the role of CXCL10 in MSC migration towards the HI-injured brain. METHODS: HI was induced in male and female 9-day-old C57BL/6 mice followed by intranasal MSC treatment at day 10 or 17 post-HI. CXCL10 protein levels, PKH26-labeled MSCs and lesion size were assessed by ELISA, immunofluorescent imaging and MAP2 staining respectively. At day 17 post-HI, when CXCL10 levels were reduced, intracranial CXCL10 injection and intranasal PKH26-labeled MSC administration were combined to assess CXCL10-guided MSC migration. MSC treatment efficacy was evaluated after 18 days, measuring lesion size, motor outcome (cylinder rearing task), glial scarring (GFAP staining) and neuronal density (NeuN staining) around the lesion. Expression of the receptor for CXCL10, i.e. CXCR3, on MSCs was confirmed by qPCR and Western Blot. Moreover, CXCL10-guided MSC migration was assessed through an in vitro transwell migration assay. RESULTS: Intranasal MSC treatment at day 17 post-HI did not reduce lesion size in contrast to earlier treatment timepoints. Cerebral CXCL10 levels were significantly decreased at 17 days versus 10 days post-HI and correlated with reduced MSC migration towards the brain. In vitro experiments demonstrated that CXCR3 receptor inhibition prevented CXCL10-guided migration of MSCs. Intracranial CXCL10 injection at day 17 post-HI significantly increased the number of MSCs reaching the lesion which was accompanied by repair of the HI lesion as measured by reduced lesion size and glial scarring, and an increased number of neurons around the lesion. CONCLUSIONS: This study underscores the crucial role of the chemoattractant CXCL10 in guiding MSCs to the HI lesion after intranasal administration. Strategies to enhance CXCR3-mediated migration of MSCs may improve the efficacy of MSC therapy or extend its regenerative therapeutic window.

Neonatal brain injury unravels transcriptional and signaling changes underlying the reactivation of cortical progenitors.

Germinal activity persists throughout life within the ventricular-subventricular zone (V-SVZ) of the postnatal forebrain due to the presence of neural stem cells (NSCs). Accumulating evidence points to a recruitment for these cells following early brain injuries and suggests their amenability to manipulations. We used chronic hypoxia as a rodent model of early brain injury to investigate the reactivation of cortical progenitors at postnatal times. Our results reveal an increased proliferation and production of glutamatergic progenitors within the dorsal V-SVZ. Fate mapping of V-SVZ NSCs demonstrates their contribution to de novo cortical neurogenesis. Transcriptional analysis of glutamatergic progenitors shows parallel changes in methyltransferase 14 (Mettl14) and Wnt/ß-catenin signaling. In agreement, manipulations through genetic and pharmacological activation of Mettl14 and the Wnt/ß-catenin pathway, respectively, induce neurogenesis and promote newly-formed cell maturation. Finally, labeling of young adult NSCs demonstrates that pharmacological NSC activation has no adverse effects on the reservoir of V-SVZ NSCs and on their germinal activity.

Transcriptomic and morphological maturation of human astrocytes in cerebral organoids.

Cerebral organoids (CerOrgs) derived from human induced pluripotent stem cells (iPSCs) are a valuable tool to study human astrocytes and their interaction with neurons and microglia. The timeline of astrocyte development and maturation in this model is currently unknown and this limits the value and applicability of the model. Therefore, we generated CerOrgs from three healthy individuals and assessed astrocyte maturation after 5, 11, 19, and 37 weeks in culture. At these four time points, the astrocyte lineage was isolated based on the expression of integrin subunit alpha 6 (ITGA6). Based on the transcriptome of the isolated ITGA6-positive cells, astrocyte development started between 5 and 11 weeks in culture and astrocyte maturation commenced after 11 weeks in culture. After 19 weeks in culture, the ITGA6-positive astrocytes had the highest expression of human mature astrocyte genes, and the predicted functional properties were related to brain homeostasis. After 37 weeks in culture, a subpopulation of ITGA6-negative astrocytes appeared, highlighting the heterogeneity within the astrocytes. The morphology shifted from an elongated progenitor-like morphology to the typical bushy astrocyte morphology. Based on the morphological properties, predicted functional properties, and the similarities with the human mature astrocyte transcriptome, we concluded that ITGA6-positive astrocytes have developed optimally in 19-week-old CerOrgs.

Single-cell profiling of human subventricular zone progenitors identifies SFRP1 as a target to re-activate progenitors.

Following the decline of neurogenesis at birth, progenitors of the subventricular zone (SVZ) remain mostly in a quiescent state in the adult human brain. The mechanisms that regulate this quiescent state are still unclear. Here, we isolate CD271+ progenitors from the aged human SVZ for single-cell RNA sequencing analysis. Our transcriptome data reveal the identity of progenitors of the aged human SVZ as late oligodendrocyte progenitor cells. We identify the Wnt pathway antagonist SFRP1 as a possible signal that promotes quiescence of progenitors from the aged human SVZ. Administration of WAY-316606, a small molecule that inhibits SFRP1 function, stimulates activation of neural stem cells both in vitro and in vivo under homeostatic conditions. Our data unravel a possible mechanism through which progenitors of the adult human SVZ are maintained in a quiescent state and a potential target for stimulating progenitors to re-activate.

Transcriptomic and functional analysis of Aß1-42 oligomer-stimulated human monocyte-derived microglia-like cells.

Dysregulation of microglial function contributes to Alzheimer's disease (AD) pathogenesis. Several genetic and transcriptome studies have revealed microglia specific genetic risk factors, and changes in microglia expression profiles in AD pathogenesis, viz. the human-Alzheimer's microglia/myeloid (HAM) profile in AD patients and the disease-associated microglia profile (DAM) in AD mouse models. The transcriptional changes involve genes in immune and inflammatory pathways, and in pathways associated with Aß clearance. Aß oligomers have been suggested to be the initial trigger of microglia activation in AD. To study the direct response to Aß oligomers exposure, we assessed changes in gene expression in an in vitro model for microglia, the human monocyte-derived microglial-like (MDMi) cells. We confirmed the initiation of an inflammatory profile following LPS stimulation, based on increased expression of IL1B, IL6, and TNFα. In contrast, the Aß1-42 oligomers did not induce an inflammatory profile or a classical HAM profile. Interestingly, we observed a specific increase in the expression of metallothioneins in the Aß1-42 oligomer treated MDMi cells. Metallothioneins are involved in metal ion regulation, protection against reactive oxygen species, and have anti-inflammatory properties. In conclusion, our data suggests that exposure to Aß1-42 oligomers may initially trigger a protective response in vitro.

The adult human subventricular zone: partial ependymal coverage and proliferative capacity of cerebrospinal fluid.

Neurogenesis continues throughout adulthood in specialized regions of the brain. One of these regions is the subventricular zone. During brain development, neurogenesis is regulated by a complex interplay of intrinsic and extrinsic cues that control stem-cell survival, renewal and cell lineage specification. Cerebrospinal fluid (CSF) is an integral part of the neurogenic niche in development as it is in direct contact with radial glial cells, and it is important in regulating proliferation and migration. Yet, the effect of CSF on neural stem cells in the subventricular zone of the adult human brain is unknown. We hypothesized a persistent stimulating effect of ventricular CSF on neural stem cells in adulthood, based on the literature, describing bulging accumulations of subventricular cells where CSF is in direct contact with the subventricular zone. Here, we show by immunohistochemistry on post-mortem adult human subventricular zone sections that neural stem cells are in close contact with CSF via protrusions through both intact and incomplete ependymal layers. We are the first to systematically quantify subventricular glial nodules denuded of ependyma and consisting of proliferating neural stem and progenitor cells, and showed that they are present from foetal age until adulthood. Neurosphere, cell motility and differentiation assays as well as analyses of RNA expression were used to assess the effects of CSF of adult humans on primary neural stem cells and a human immortalized neural stem cell line. We show that human ventricular CSF increases proliferation and decreases motility of neural stem cells. Our results also indicate that adult CSF pushes neural stem cells from a relative quiescent to a more active state and promotes neuronal over astrocytic lineage differentiation. Thus, CSF continues to stimulate neural stem cells throughout aging.

Transcriptome and proteome profiling of neural stem cells from the human subventricular zone in Parkinson's disease.

It is currently accepted that the human brain has a limited neurogenic capacity and an impaired regenerative potential. We have previously shown the existence of CD271-expressing neural stem cells (NSCs) in the subventricular zone (SVZ) of Parkinson's disease (PD) patients, which proliferate and differentiate towards neurons and glial cells in vitro. To study the molecular profile of these NSCs in detail, we performed RNA sequencing and mass spectrometry on CD271+ NSCs isolated from human post-mortem SVZ and on homogenates of the SVZ. CD271+ cells were isolated through magnetic cell separation (MACS). We first compared the molecular profile of CD271+ NSCs to the SVZ homogenate from control donors and then compared CD271+ cells to CD11b+ microglia. These results confirmed their neural stem cell identity. Finally we compared controls and PD patients to establish a specific molecular profile of NSCs and the SVZ in PD. While our transcriptome analysis did not identify any differentially expressed genes in the SVZ between control and PD patients, our proteome analysis revealed several proteins that were differentially expressed in PD. Some of these proteins are involved in cytoskeletal organization and mitochondrial function. Transcriptome and proteome analyses of NSCs from PD revealed changes in the expression of genes and proteins involved in metabolism, transcriptional activity and cytoskeletal organization. Our data suggest that NSCs may transit into a primed-quiescent state, that is in an "alert" non-proliferative phase in PD. Our results not only confirm pathological hallmarks of PD (e.g. impaired mitochondrial function), but also show that the NSCs from SVZ undergo significant changes at both transcriptome and proteome level following PD.

Transcriptional Dysregulation in Postnatal Glutamatergic Progenitors Contributes to Closure of the Cortical Neurogenic Period.

Progenitors of cortical glutamatergic neurons (Glu progenitors) are usually thought to switch fate before birth to produce astrocytes. We used fate-mapping approaches to show that a large fraction of Glu progenitors persist in the postnatal forebrain after closure of the cortical neurogenesis period. Postnatal Glu progenitors do not accumulate during embryonal development but are produced by embryonal radial glial cells that persist after birth in the dorsal subventricular zone and continue to give rise to cortical neurons, although with low efficiency. Single-cell RNA sequencing reveals a dysregulation of transcriptional programs, which parallels changes in m6A methylation and correlates with the gradual decline in cortical neurogenesis observed in vivo. Rescuing experiments show that postnatal progenitors are partially permissive to genetic and pharmacological manipulations. Our study provides an in-depth characterization of postnatal Glu progenitors and identifies potential therapeutic targets for promoting brain repair.

Pharmacogenomic identification of small molecules for lineage specific manipulation of subventricular zone germinal activity.

Strategies for promoting neural regeneration are hindered by the difficulty of manipulating desired neural fates in the brain without complex genetic methods. The subventricular zone (SVZ) is the largest germinal zone of the forebrain and is responsible for the lifelong generation of interneuron subtypes and oligodendrocytes. Here, we have performed a bioinformatics analysis of the transcriptome of dorsal and lateral SVZ in early postnatal mice, including neural stem cells (NSCs) and their immediate progenies, which generate distinct neural lineages. We identified multiple signaling pathways that trigger distinct downstream transcriptional networks to regulate the diversity of neural cells originating from the SVZ. Next, we used a novel in silico genomic analysis, searchable platform-independent expression database/connectivity map (SPIED/CMAP), to generate a catalogue of small molecules that can be used to manipulate SVZ microdomain-specific lineages. Finally, we demonstrate that compounds identified in this analysis promote the generation of specific cell lineages from NSCs in vivo, during postnatal life and adulthood, as well as in regenerative contexts. This study unravels new strategies for using small bioactive molecules to direct germinal activity in the SVZ, which has therapeutic potential in neurodegenerative diseases.

Postnatal Neural Stem Cells: Probing Their Competence for Cortical Repair.

There is growing evidence for a tentative cellular repair in the forebrain following perinatal injuries. In this review, we present the evidences and shortcomings in this regenerative attempt. We discuss recent progress in elucidating the origin, diversity, and competence of postnatal neural stem cells/progenitor cells. Finally, we propose new strategies to recruit postnatal progenitors to generate specific subtypes of cortical neurons or oligodendrocytes, thereby allowing the development of tailor-made approaches to treat perinatal brain injuries.

Assessment of long-term safety and efficacy of intranasal mesenchymal stem cell treatment for neonatal brain injury in the mouse.

BACKGROUND: For clinical translation, we assessed whether intranasal mesenchymal stem cell (MSC) treatment after hypoxia-ischemia (HI) induces neoplasia in the brain or periphery at 14 mo. Furthermore, the long-term effects of MSCs on behavior and lesion size were determined. METHOD: HI was induced in 9-d-old mice. Pups received an intranasal administration of 0.5 × 10(6) MSCs or vehicle at 10 d post-HI. Full macroscopical and microscopical pathological analysis of 39 organs per mouse was performed. Sensorimotor behavior was assessed in the cylinder-rearing test at 10 d, 28 d, 6 mo, and 9 mo. Cognition was measured with the novel object recognition test at 3 and 14 mo post-HI. Lesion size was determined by analyzing mouse-anti-microtubule-associated protein 2 (MAP2) and mouse-anti-myelin basic protein (MBP) staining at 5 wk and 14 mo. RESULTS: At 14 mo post-HI, we did not observe any neoplasia in the nasal turbinates, brain, or other organs of HI mice treated with MSCs. Furthermore, our results show that MSC-induced improvement of sensorimotor and cognitive function is long lasting. In contrast, HI-vehicle mice showed severe behavioral impairment. Recovery of MAP2- and MBP-positive area lasted up to 14 mo following MSC treatment. CONCLUSION: Our results provide strong evidence of the long-term safety and positive effects of MSC treatment following neonatal HI in mice.

Intranasal administration of human MSC for ischemic brain injury in the mouse: in vitro and in vivo neuroregenerative functions.

Intranasal treatment with C57BL/6 MSCs reduces lesion volume and improves motor and cognitive behavior in the neonatal hypoxic-ischemic (HI) mouse model. In this study, we investigated the potential of human MSCs (hMSCs) to treat HI brain injury in the neonatal mouse. Assessing the regenerative capacity of hMSCs is crucial for translation of our knowledge to the clinic. We determined the neuroregenerative potential of hMSCs in vitro and in vivo by intranasal administration 10 d post-HI in neonatal mice. HI was induced in P9 mouse pups. 1×10(6) or 2×10(6) hMSCs were administered intranasally 10 d post-HI. Motor behavior and lesion volume were measured 28 d post-HI. The in vitro capacity of hMSCs to induce differentiation of mouse neural stem cell (mNSC) was determined using a transwell co-culture differentiation assay. To determine which chemotactic factors may play a role in mediating migration of MSCs to the lesion, we performed a PCR array on 84 chemotactic factors 10 days following sham-operation, and at 10 and 17 days post-HI. Our results show that 2×10(6) hMSCs decrease lesion volume, improve motor behavior, and reduce scar formation and microglia activity. Moreover, we demonstrate that the differentiation assay reflects the neuroregenerative potential of hMSCs in vivo, as hMSCs induce mNSCs to differentiate into neurons in vitro. We also provide evidence that the chemotactic factor CXCL10 may play an important role in hMSC migration to the lesion site. This is suggested by our finding that CXCL10 is significantly upregulated at 10 days following HI, but not at 17 days after HI, a time when MSCs no longer reach the lesion when given intranasally. The results described in this work also tempt us to contemplate hMSCs not only as a potential treatment option for neonatal encephalopathy, but also for a plethora of degenerative and traumatic injuries of the nervous system.

Intranasally administered mesenchymal stem cells promote a regenerative niche for repair of neonatal ischemic brain injury.

Previous work from our group has shown that intranasal MSC-treatment decreases lesion volume and improves motor and cognitive behavior after hypoxic-ischemic (HI) brain damage in neonatal mice. Our aim was to determine the kinetics of MSC migration after intranasal administration, and the early effects of MSCs on neurogenic processes and gliosis at the lesion site. HI brain injury was induced in 9-day-old mice and MSCs were administered intranasally at 10days post-HI. The kinetics of MSC migration were investigated by immunofluorescence and MRI analysis. BDNF and NGF gene expression was determined by qPCR analysis following MSC co-culture with HI brain extract. Nestin, Doublecortin, NeuN, GFAP, Iba-1 and M1/M2 phenotypic expression was assessed over time. MRI and immunohistochemistry analyses showed that MSCs reach the lesion site already within 2h after intranasal administration. At 12h after administration the number of MSCs at the lesion site peaks and decreases significantly at 72h. The number of DCX(+) cells increased 1 to 3days after MSC administration in the SVZ. At the lesion, GFAP(+)/nestin(+) and DCX(+) expression increased 3 to 5days after MSC-treatment. The number of NeuN(+) cells increased within 5days, leading to a dramatic regeneration of the somatosensory cortex and hippocampus at 18days after intranasal MSC administration. Interestingly, MSCs expressed significantly more BDNF gene when exposed to HI brain extract in vitro. Furthermore, MSC-treatment resulted in the resolution of the glial scar surrounding the lesion, represented by a decrease in reactive astrocytes and microglia and polarization of microglia towards the M2 phenotype. In view of the current lack of therapeutic strategies, we propose that intranasal MSC administration is a powerful therapeutic option through its functional repair of the lesion represented by regeneration of the cortical and hippocampal structure and decrease of gliosis.

The endogenous regenerative capacity of the damaged newborn brain: boosting neurogenesis with mesenchymal stem cell treatment.

Neurogenesis continues throughout adulthood. The neurogenic capacity of the brain increases after injury by, e.g., hypoxia-ischemia. However, it is well known that in many cases brain damage does not resolve spontaneously, indicating that the endogenous regenerative capacity of the brain is insufficient. Neonatal encephalopathy leads to high mortality rates and long-term neurologic deficits in babies worldwide. Therefore, there is an urgent need to develop more efficient therapeutic strategies. The latest findings indicate that stem cells represent a novel therapeutic possibility to improve outcome in models of neonatal encephalopathy. Transplanted stem cells secrete factors that stimulate and maintain neurogenesis, thereby increasing cell proliferation, neuronal differentiation, and functional integration. Understanding the molecular and cellular mechanisms underlying neurogenesis after an insult is crucial for developing tools to enhance the neurogenic capacity of the brain. The aim of this review is to discuss the endogenous capacity of the neonatal brain to regenerate after a cerebral ischemic insult. We present an overview of the molecular and cellular mechanisms underlying endogenous regenerative processes during development as well as after a cerebral ischemic insult. Furthermore, we will consider the potential to use stem cell transplantation as a means to boost endogenous neurogenesis and restore brain function.

Intranasal mesenchymal stem cell treatment for neonatal brain damage: long-term cognitive and sensorimotor improvement.

Mesenchymal stem cell (MSC) administration via the intranasal route could become an effective therapy to treat neonatal hypoxic-ischemic (HI) brain damage. We analyzed long-term effects of intranasal MSC treatment on lesion size, sensorimotor and cognitive behavior, and determined the therapeutic window and dose response relationships. Furthermore, the appearance of MSCs at the lesion site in relation to the therapeutic window was examined. Nine-day-old mice were subjected to unilateral carotid artery occlusion and hypoxia. MSCs were administered intranasally at 3, 10 or 17 days after hypoxia-ischemia (HI). Motor, cognitive and histological outcome was investigated. PKH-26 labeled cells were used to localize MSCs in the brain. We identified 0.5 × 10(6) MSCs as the minimal effective dose with a therapeutic window of at least 10 days but less than 17 days post-HI. A single dose was sufficient for a marked beneficial effect. MSCs reach the lesion site within 24 h when given 3 or 10 days after injury. However, no MSCs were detected in the lesion when administered 17 days following HI. We also show for the first time that intranasal MSC treatment after HI improves cognitive function. Improvement of sensorimotor function and histological outcome was maintained until at least 9 weeks post-HI. The capacity of MSCs to reach the lesion site within 24 h after intranasal administration at 10 days but not at 17 days post-HI indicates a therapeutic window of at least 10 days. Our data strongly indicate that intranasal MSC treatment may become a promising non-invasive therapeutic tool to effectively reduce neonatal encephalopathy.