Human monocyte subtype expression of neuroinflammation and regeneration-related genes is linked to age and sex.

Stroke is a leading cause of disability and the third cause of death. The immune system plays an essential role in post-stroke recovery. After an ischemic stroke, monocytes infiltrate the injured brain tissue and can exacerbate or mitigate the damage. Ischemic stroke is more prevalent in the aged population, and the aging brain exhibits an altered immune response. There are also sex disparities in ischemic stroke incidence, outcomes, and recovery, and these differences may be hormone-driven and determined by genetic and epigenetic factors. Here, we studied whether human peripheral blood monocyte subtype (classical, intermediate, and non-classical) expression of neuronal inflammation- and regeneration-related genes depends on age and sex. A FACS analysis of blood samples from 44 volunteers (male and female, aged 28 to 98) showed that in contrast to other immune cells, the proportion of natural killer cells increased in females. The proportion of B-cells decreased in both sexes with age, and subtypes of monocytes were not linked to age or sex. Gene expression analysis by qPCR identified several genes differentially correlating with age and sex within different monocyte subtypes. Interestingly, ANXA1 and CD36 showed a consistent increase with aging in all monocytes, specifically in intermediate (CD36) and intermediate and non-classical (ANXA1) subtypes. Other genes (IL-1ß, S100A8, TNFα, CD64, CD33, TGFß1, TLR8, CD91) were differentially changed in monocyte subtypes with increased aging. Most age-dependent gene changes were differentially expressed in female monocytes. Our data shed light on the nuanced interplay of age and sex in shaping the expression of inflammation- and regeneration-related genes within distinct monocyte subtypes. Understanding these dynamics could pave the way for targeted interventions and personalized approaches in post-stroke care, particularly for the aging population and individuals of different sexes.

LINE-1 retrotransposons drive human neuronal transcriptome complexity and functional diversification.

The genetic mechanisms underlying the expansion in size and complexity of the human brain remain poorly understood. Long interspersed nuclear element-1 (L1) retrotransposons are a source of divergent genetic information in hominoid genomes, but their importance in physiological functions and their contribution to human brain evolution are largely unknown. Using multiomics profiling, we here demonstrate that L1 promoters are dynamically active in the developing and the adult human brain. L1s generate hundreds of developmentally regulated and cell type-specific transcripts, many that are co-opted as chimeric transcripts or regulatory RNAs. One L1-derived long noncoding RNA, LINC01876, is a human-specific transcript expressed exclusively during brain development. CRISPR interference silencing of LINC01876 results in reduced size of cerebral organoids and premature differentiation of neural progenitors, implicating L1s in human-specific developmental processes. In summary, our results demonstrate that L1-derived transcripts provide a previously undescribed layer of primate- and human-specific transcriptome complexity that contributes to the functional diversification of the human brain.

Oligodendrocytes in human induced pluripotent stem cell-derived cortical grafts remyelinate adult rat and human cortical neurons.

Neuronal loss and axonal demyelination underlie long-term functional impairments in patients affected by brain disorders such as ischemic stroke. Stem cell-based approaches reconstructing and remyelinating brain neural circuitry, leading to recovery, are highly warranted. Here, we demonstrate the in vitro and in vivo production of myelinating oligodendrocytes from a human induced pluripotent stem cell (iPSC)-derived long-term neuroepithelial stem (lt-NES) cell line, which also gives rise to neurons with the capacity to integrate into stroke-injured, adult rat cortical networks. Most importantly, the generated oligodendrocytes survive and form myelin-ensheathing human axons in the host tissue after grafting onto adult human cortical organotypic cultures. This lt-NES cell line is the first human stem cell source that, after intracerebral delivery, can repair both injured neural circuitries and demyelinated axons. Our findings provide supportive evidence for the potential future use of human iPSC-derived cell lines to promote effective clinical recovery following brain injuries.

MafB-dependent neurotransmitter signaling promotes ß cell migration in the developing pancreas.

Hormone secretion from pancreatic islets is essential for glucose homeostasis, and loss or dysfunction of islet cells is a hallmark of type 2 diabetes. Maf transcription factors are crucial for establishing and maintaining adult endocrine cell function. However, during pancreas development, MafB is not only expressed in insulin- and glucagon-producing cells, but also in Neurog3+ endocrine progenitor cells, suggesting additional functions in cell differentiation and islet formation. Here, we report that MafB deficiency impairs ß cell clustering and islet formation, but also coincides with loss of neurotransmitter and axon guidance receptor gene expression. Moreover, the observed loss of nicotinic receptor gene expression in human and mouse ß cells implied that signaling through these receptors contributes to islet cell migration/formation. Inhibition of nicotinic receptor activity resulted in reduced ß cell migration towards autonomic nerves and impaired ß cell clustering. These findings highlight a novel function of MafB in controlling neuronal-directed signaling events required for islet formation.

Adult hippocampal neurogenesis and social behavioural deficits in the R451C Neuroligin3 mouse model of autism are reverted by the antidepressant fluoxetine.

Neuron generation persists throughout life in the hippocampus but is altered in animal models of neurological and neuropsychiatric diseases, suggesting that disease-associated decline in cognitive and emotional hippocampal-dependent behaviours might be functionally linked with dysregulation of postnatal neurogenesis. Depletion of the adult neural stem/progenitor cell (NSPCs) pool and neurogenic decline have been recently described in mice expressing synaptic susceptibility genes associated with autism spectrum disorder (ASDs). To gain further insight into mechanisms regulating neurogenesis in mice carrying mutations in synaptic genes related to monogenic ASDs, we used the R451C Neuroligin3 knock-in (Nlgn3 KI) mouse, which is characterized by structural brain abnormalities, deficits in synaptic functions and reduced sociability. We show that the number of adult-born neurons, but not the size of the NSPC pool, was reduced in the ventral dentate gyrus in knock-in mice. Notably, this neurogenic decline was rescued by daily injecting mice with 10 mg/Kg of the antidepressant fluoxetine for 20 consecutive days. Sustained treatment also improved KI mice's sociability and increased the number of c-Fos active adult-born neurons, compared with vehicle-injected KI mice. Our study uncovers neurogenesis-mediated alterations in the brain of R451C KI mouse, showing that the R451C Nlgn3 mutation leads to lasting, albeit pharmacologically reversible, changes in the brain, affecting neuron formation in the adult hippocampus. Our results suggest that fluoxetine can ameliorate social behaviour in KI mice, at least in part, by rescuing adult hippocampal neurogenesis, which may be relevant for the pharmacological treatment of ASDs.

Long-term calcium imaging reveals functional development in hiPSC-derived cultures comparable to human but not rat primary cultures.

Models for human brain-oriented research are often established on primary cultures from rodents, which fails to recapitulate cellular specificity and molecular cues of the human brain. Here we investigated whether neuronal cultures derived from human induced pluripotent stem cells (hiPSCs) feature key advantages compared with rodent primary cultures. Using calcium fluorescence imaging, we tracked spontaneous neuronal activity in hiPSC-derived, human, and rat primary cultures and compared their dynamic and functional behavior as they matured. We observed that hiPSC-derived cultures progressively changed upon development, exhibiting gradually richer activity patterns and functional traits. By contrast, rat primary cultures were locked in the same dynamic state since activity onset. Human primary cultures exhibited features in between hiPSC-derived and rat primary cultures, although traits from the former predominated. Our study demonstrates that hiPSC-derived cultures are excellent models to investigate development in neuronal assemblies, a hallmark for applications that monitor alterations caused by damage or neurodegeneration.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation.

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Simulated Microgravity Modulates Focal Adhesion Gene Expression in Human Neural Stem Progenitor Cells.

We analyzed the morphology and the transcriptomic changes of human neural stem progenitor cells (hNSPCs) grown on laminin in adherent culture conditions and subjected to simulated microgravity for different times in a random positioning machine apparatus. Low-cell-density cultures exposed to simulated microgravity for 24 h showed cell aggregate formation and significant modulation of several genes involved in focal adhesion, cytoskeleton regulation, and cell cycle control. These effects were much more limited in hNSPCs cultured at high density in the same conditions. We also found that some of the genes modulated upon exposure to simulated microgravity showed similar changes in hNSPCs grown without laminin in non-adherent culture conditions under normal gravity. These results suggest that reduced gravity counteracts the interactions of cells with the extracellular matrix, inducing morphological and transcriptional changes that can be observed in low-density cultures.

Human stem cell-derived GABAergic neurons functionally integrate into human neuronal networks.

Gamma-aminobutyric acid (GABA)-releasing interneurons modulate neuronal network activity in the brain by inhibiting other neurons. The alteration or absence of these cells disrupts the balance between excitatory and inhibitory processes, leading to neurological disorders such as epilepsy. In this regard, cell-based therapy may be an alternative therapeutic approach. We generated light-sensitive human embryonic stem cell (hESC)-derived GABAergic interneurons (hdIN) and tested their functionality. After 35 days in vitro (DIV), hdINs showed electrophysiological properties and spontaneous synaptic currents comparable to mature neurons. In co-culture with human cortical neurons and after transplantation (AT) into human brain tissue resected from patients with drug-resistant epilepsy, light-activated channelrhodopsin-2 (ChR2) expressing hdINs induced postsynaptic currents in human neurons, strongly suggesting functional efferent synapse formation. These results provide a proof-of-concept that hESC-derived neurons can integrate and modulate the activity of a human host neuronal network. Therefore, this study supports the possibility of precise temporal control of network excitability by transplantation of light-sensitive interneurons.

Pericyte-derived fibrotic scarring is conserved across diverse central nervous system lesions.

Fibrotic scar tissue limits central nervous system regeneration in adult mammals. The extent of fibrotic tissue generation and distribution of stromal cells across different lesions in the brain and spinal cord has not been systematically investigated in mice and humans. Furthermore, it is unknown whether scar-forming stromal cells have the same origin throughout the central nervous system and in different types of lesions. In the current study, we compared fibrotic scarring in human pathological tissue and corresponding mouse models of penetrating and non-penetrating spinal cord injury, traumatic brain injury, ischemic stroke, multiple sclerosis and glioblastoma. We show that the extent and distribution of stromal cells are specific to the type of lesion and, in most cases, similar between mice and humans. Employing in vivo lineage tracing, we report that in all mouse models that develop fibrotic tissue, the primary source of scar-forming fibroblasts is a discrete subset of perivascular cells, termed type A pericytes. Perivascular cells with a type A pericyte marker profile also exist in the human brain and spinal cord. We uncover type A pericyte-derived fibrosis as a conserved mechanism that may be explored as a therapeutic target to improve recovery after central nervous system lesions.

Neuronal Replacement in Stem Cell Therapy for Stroke: Filling the Gap.

Stem cell therapy using human skin-derived neural precursors holds much promise for the treatment of stroke patients. Two main mechanisms have been proposed to give rise to the improved recovery in animal models of stroke after transplantation of these cells. First, the so called by-stander effect, which could modulate the environment during early phases after brain tissue damage, resulting in moderate improvements in the outcome of the insult. Second, the neuronal replacement and functional integration of grafted cells into the impaired brain circuitry, which will result in optimum long-term structural and functional repair. Recently developed sophisticated research tools like optogenetic control of neuronal activity and rabies virus monosynaptic tracing, among others, have made it possible to provide solid evidence about the functional integration of grafted cells and its contribution to improved recovery in animal models of brain damage. Moreover, previous clinical trials in patients with Parkinson's Disease represent a proof of principle that stem cell-based neuronal replacement could work in humans. Our studies with in vivo and ex vivo transplantation of human skin-derived cells neurons in animal model of stroke and organotypic cultures of adult human cortex, respectively, also support the hypothesis that human somatic cells reprogrammed into neurons can get integrated in the human lesioned neuronal circuitry. In the present short review, we summarized our data and recent studies from other groups supporting the above hypothesis and opening new avenues for development of the future clinical applications.

NGN2 mmRNA-Based Transcriptional Programming in Microfluidic Guides hiPSCs Toward Neural Fate With Multiple Identities.

Recent advancements in cell engineering have succeeded in manipulating cell identity with the targeted overexpression of specific cell fate determining transcription factors in a process named transcriptional programming. Neurogenin2 (NGN2) is sufficient to instruct pluripotent stem cells (PSCs) to acquire a neuronal identity when delivered with an integrating system, which arises some safety concerns for clinical applications. A non-integrating system based on modified messenger RNA (mmRNA) delivery method, represents a valuable alternative to lentiviral-based approaches. The ability of NGN2 mmRNA to instruct PSC fate change has not been thoroughly investigated yet. Here we aimed at understanding whether the use of an NGN2 mmRNA-based approach combined with a miniaturized system, which allows a higher transfection efficiency in a cost-effective system, is able to drive human induced PSCs (hiPSCs) toward the neuronal lineage. We show that NGN2 mRNA alone is able to induce cell fate conversion. Surprisingly, the outcome cell population accounts for multiple phenotypes along the neural development trajectory. We found that this mixed population is mainly constituted by neural stem cells (45% ± 18 PAX6 positive cells) and neurons (38% ± 8 ßIIITUBULIN positive cells) only when NGN2 is delivered as mmRNA. On the other hand, when the delivery system is lentiviral-based, both providing a constant expression of NGN2 or only a transient pulse, the outcome differentiated population is formed by a clear majority of neurons (88% ± 1 ßIIITUBULIN positive cells). Altogether, our data confirm the ability of NGN2 to induce neuralization in hiPSCs and opens a new point of view in respect to the delivery system method when it comes to transcriptional programming applications.

New Mechanistic Insights, Novel Treatment Paradigms, and Clinical Progress in Cerebrovascular Diseases.

The past decade has brought tremendous progress in diagnostic and therapeutic options for cerebrovascular diseases as exemplified by the advent of thrombectomy in ischemic stroke, benefitting a steeply increasing number of stroke patients and potentially paving the way for a renaissance of neuroprotectants. Progress in basic science has been equally impressive. Based on a deeper understanding of pathomechanisms underlying cerebrovascular diseases, new therapeutic targets have been identified and novel treatment strategies such as pre- and post-conditioning methods were developed. Moreover, translationally relevant aspects are increasingly recognized in basic science studies, which is believed to increase their predictive value and the relevance of obtained findings for clinical application.This review reports key results from some of the most remarkable and encouraging achievements in neurovascular research that have been reported at the 10th International Symposium on Neuroprotection and Neurorepair. Basic science topics discussed herein focus on aspects such as neuroinflammation, extracellular vesicles, and the role of sex and age on stroke recovery. Translational reports highlighted endovascular techniques and targeted delivery methods, neurorehabilitation, advanced functional testing approaches for experimental studies, pre-and post-conditioning approaches as well as novel imaging and treatment strategies. Beyond ischemic stroke, particular emphasis was given on activities in the fields of traumatic brain injury and cerebral hemorrhage in which promising preclinical and clinical results have been reported. Although the number of neutral outcomes in clinical trials is still remarkably high when targeting cerebrovascular diseases, we begin to evidence stepwise but continuous progress towards novel treatment options. Advances in preclinical and translational research as reported herein are believed to have formed a solid foundation for this progress.

SmartFlareTM is a reliable method for assessing mRNA expression in single neural stem cells.

BACKGROUND: One of the most challenging tasks of modern biology concerns the real-time tracking and quantification of mRNA expression in living cells. On this matter, a novel platform called SmartFlareTM has taken advantage of fluorophore-linked nanoconstructs for targeting RNA transcripts. Although fluorescence emission does not account for the spatial mRNA distribution, NanoFlare technology has grown a range of theranostic applications starting from detecting biomarkers related to diseases, such as cancer, neurodegenerative pathologies or embryonic developmental disorders. AIM: To investigate the potential of SmartFlareTM in determining time-dependent mRNA expression of prominin 1 (CD133) and octamer-binding transcription factor 4 (OCT4) in single living cells through differentiation. METHODS: Brain fragments from the striatum of aborted human fetuses aged 8 wk postconception were processed to obtain neurospheres. For the in vitro differentiation, neurospheres were gently dissociated with Accutase solution. Single cells were resuspended in a basic medium enriched with fetal bovine serum, plated on poly-L-lysine-coated glass coverslips, and grown in a lapse of time from 1 to 4 wk. Live cell mRNA detection was performed using SmartFlareTM probes (CD133, Oct4, Actin, and Scramble). All the samples were incubated at 37 °C for 24 h. For nuclear staining, Hoechst 33342 was added. SmartFlareTM CD133- and OCT4-specific fluorescence signal was assessed using a semiquantitative visual approach, taking into account the fluorescence intensity and the number of labeled cells. RESULTS: In agreement with previous PCR experiments, a unique expression trend was observed for CD133 and OCT4 genes until 7 d in vitro (DIV). Fluorescence resulted in a mixture of diffuse cytoplasmic and spotted-like pattern, also detectable in the contacting neural branches. From 15 to 30 DIV, only few cells showed a scattered fluorescent pattern, in line with the differentiation progression and coherent with mRNA downregulation of these stemness-related genes. CONCLUSION: SmartFlareTM appears to be a reliable, easy-to-handle tool for investigating CD133 and OCT4 expression in a neural stem cell model, preserving cell biological properties in anticipation of downstream experiments.

Hypoxia inducible factor-2α importance for migration, proliferation, and self-renewal of trunk neural crest cells.

BACKGROUND: The neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)-2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development. RESULTS: Here, we show that HIF-2α is expressed in trunk neural crest cells of human, murine, and avian embryos. Knockdown as well as overexpression of HIF-2α in vivo causes developmental delays, induces proliferation, and self-renewal capacity of neural crest cells while decreasing the proportion of neural crest cells that migrate ventrally to sympathoadrenal sites. Reflecting the in vivo phenotype, transcriptome changes after loss of HIF-2α reveal enrichment of genes associated with cancer, invasion, epithelial-to-mesenchymal transition, and growth arrest. CONCLUSIONS: Taken together, these results suggest that expression levels of HIF-2α must be strictly controlled during normal trunk neural crest development and that dysregulated levels affects several important features connected to stemness, migration, and development.

Grafted human pluripotent stem cell-derived cortical neurons integrate into adult human cortical neural circuitry.

Several neurodegenerative diseases cause loss of cortical neurons, leading to sensory, motor, and cognitive impairments. Studies in different animal models have raised the possibility that transplantation of human cortical neuronal progenitors, generated from pluripotent stem cells, might be developed into a novel therapeutic strategy for disorders affecting cerebral cortex. For example, we have shown that human long-term neuroepithelial-like stem (lt-NES) cell-derived cortical neurons, produced from induced pluripotent stem cells and transplanted into stroke-injured adult rat cortex, improve neurological deficits and establish both afferent and efferent morphological and functional connections with host cortical neurons. So far, all studies with human pluripotent stem cell-derived neurons have been carried out using xenotransplantation in animal models. Whether these neurons can integrate also into adult human brain circuitry is unknown. Here, we show that cortically fated lt-NES cells, which are able to form functional synaptic networks in cell culture, differentiate to mature, layer-specific cortical neurons when transplanted ex vivo onto organotypic cultures of adult human cortex. The grafted neurons are functional and establish both afferent and efferent synapses with adult human cortical neurons in the slices as evidenced by immuno-electron microscopy, rabies virus retrograde monosynaptic tracing, and whole-cell patch-clamp recordings. Our findings provide the first evidence that pluripotent stem cell-derived neurons can integrate into adult host neural networks also in a human-to-human grafting situation, thereby supporting their potential future clinical use to promote recovery by neuronal replacement in the patient's diseased brain.

Poly(ester amide) microspheres are efficient vehicles for long-term intracerebral growth factor delivery and improve functional recovery after stroke.

Growth factors promote plasticity in injured brain and improve impaired functions. For clinical application, efficient approaches for growth factor delivery into the brain are necessary. Poly(ester amide) (PEA)-derived microspheres (MS) could serve as vehicles due to their thermal and mechanical properties, biocompatibility and biodegradability. Vascular endothelial growth factor (VEGF) exerts both vascular and neuronal actions, making it suitable to stimulate post-stroke recovery. Here, PEA (composed of adipic acid, L-phenyl-alanine and 1,4-butanediol) MS were loaded with VEGF and injected intracerebrally in mice subjected to cortical stroke. Loaded MS provided sustained release of VEGF in vitro and, after injection, biologically active VEGF was released long-term, as evidenced by high VEGF immunoreactivity, increased VEGF tissue levels, and higher vessel density and more NG2+ cells in injured hemisphere of animals with VEGF-loaded as compared to non-loaded MS. Loaded MS gave rise to more rapid recovery of neurological score. Both loaded and non-loaded MS induced improvement in neurological score and adhesive removal test, probably due to anti-inflammatory action. In summary, grafted PEA MS can act as efficient vehicles, with anti-inflammatory action, for long-term delivery of growth factors into injured brain. Our data suggest PEA MS as a new tool for neurorestorative approaches with therapeutic potential.

Blocking Notch-Signaling Increases Neurogenesis in the Striatum after Stroke.

Stroke triggers neurogenesis in the striatum in mice, with new neurons deriving in part from the nearby subventricular zone and in part from parenchymal astrocytes. The initiation of neurogenesis by astrocytes within the striatum is triggered by reduced Notch-signaling, and blocking this signaling pathway by deletion of the gene encoding the obligate Notch coactivator Rbpj is sufficient to activate neurogenesis by striatal astrocytes in the absence of an injury. Here we report that blocking Notch-signaling in stroke increases the neurogenic response to stroke 3.5-fold in mice. Deletion of Rbpj results in the recruitment of a larger number of parenchymal astrocytes to neurogenesis and over larger areas of the striatum. These data suggest inhibition of Notch-signaling as a potential translational strategy to promote neuronal regeneration after stroke.