Microglia undergo disease-associated transcriptional activation and CX3C motif chemokine receptor 1 expression regulates neurogenesis in the aged brain.

Adult neurogenesis continues throughout life but declines dramatically with age and in neurodegenerative disorders such as Alzheimer's disease. In parallel, microglia become activated resulting in chronic inflammation in the aged brain. A unique type of microglia, suggested to support neurogenesis, exists in the subventricular zone (SVZ), but little is known how they are affected by aging. We analyzed the transcriptome of aging microglia and identified a unique neuroprotective activation profile in aged SVZ microglia, which is partly shared with disease-associated microglia (DAM). CX3C motif chemokine receptor 1 (CX3CR1) is characteristically expressed by brain microglia where it directs migration to targets for phagocytosis. We show that Cx3cr1 expression, as in DAM, is downregulated in old SVZ microglia and that heterozygous Cx3cr1 mice have increased proliferation and neuroblast number in the aged SVZ but not in the dentate gyrus, identifying CX3CR1 signaling as a novel age and brain region-specific regulator of neurogenesis.

Astrocyte dysfunction and neuronal network hyperactivity in a CRISPR engineered pluripotent stem cell model of frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most prevalent type of early-onset dementia and up to 40% of cases are familial forms. One of the genes mutated in patients is CHMP2B, which encodes a protein found in a complex important for maturation of late endosomes, an essential process for recycling membrane proteins through the endolysosomal system. Here, we have generated a CHMP2B-mutated human embryonic stem cell line using genome editing with the purpose to create a human in vitro FTD disease model. To date, most studies have focused on neuronal alterations; however, we present a new co-culture system in which neurons and astrocytes are independently generated from human embryonic stem cells and combined in co-cultures. With this approach, we have identified alterations in the endolysosomal system of FTD astrocytes, a higher capacity of astrocytes to uptake and respond to glutamate, and a neuronal network hyperactivity as well as excessive synchronization. Overall, our data indicates that astrocyte alterations precede neuronal impairments and could potentially trigger neuronal network changes, indicating the important and specific role of astrocytes in disease development.

Transcription Factor-Forced Astrocytic Differentiation Impairs Human Glioblastoma Growth In Vitro and In Vivo.

Direct cellular reprogramming has recently gained attention of cancer researchers for the possibility to convert undifferentiated cancer cells into more differentiated, postmitotic cell types. While a few studies have attempted reprogramming of glioblastoma (GBM) cells toward a neuronal fate, this approach has not yet been used to induce differentiation into other lineages and in vivo data on reduction in tumorigenicity are limited. Here, we employ cellular reprogramming to induce astrocytic differentiation as a therapeutic approach in GBM. To this end, we overexpressed key transcriptional regulators of astroglial development in human GBM and GBM stem cell lines. Treated cells undergo a remarkable shift in structure, acquiring an astrocyte-like morphology with star-shaped bodies and radial branched processes. Differentiated cells express typical glial markers and show a marked decrease in their proliferative state. In addition, forced differentiation induces astrocytic functions such as induced calcium transients and ability to respond to inflammatory stimuli. Most importantly, forced differentiation substantially reduces tumorigenicity of GBM cells in an in vivo xenotransplantation model. The current study capitalizes on cellular plasticity with a novel application in cancer. We take advantage of the similarity between neural developmental processes and cancer hierarchy to mitigate, if not completely abolish, the malignant nature of tumor cells and pave the way for new intervention strategies.

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.

Past, Present, and Future of Direct Cell Reprogramming.

Budding off from the broader developmental biology and stem cell research fields, cellular reprogramming is now established as a prominent discipline in its own right. Direct cell reprogramming is defined as the cell fate conversion of a somatic cell toward another identity without a pluripotent intermediate state. In addition to the opportunity for mechanistic dissection of lineage commitment in human cells, the field offer the promise of diverse applications such as for disease modeling, cell replacement therapy, regenerative medicine, and immunotherapy that have recently spurred innovation and out of the box thinking to unleash the potential of cellular plasticity.

Transcription factor-based direct conversion of human fibroblasts to functional astrocytes.

Astrocytes are emerging key players in neurological disorders. However, their role in disease etiology is poorly understood owing to inaccessibility of primary human astrocytes. Pluripotent stem cell-derived cells fail to mimic age and due to their clonal origin do not mimic genetic heterogeneity of patients. In contrast, direct conversion constitutes an attractive approach to generate human astrocytes that capture age and genetic diversity. We describe efficient direct conversion of human fibroblasts to functional induced astrocytes (iAs). Expression of the minimal combination Sox9 and Nfib generates iAs with molecular, phenotypic, and functional properties resembling primary human astrocytes. iAs could be obtained by conversion of fibroblasts covering the entire human lifespan. Importantly, iAs supported function of induced neurons obtained through direct conversion from the same fibroblast population. Fibroblast-derived iAs will become a useful tool to elucidate the biology of astrocytes and complement current in vitro models for studies of late-onset neurological disorders.

Transcription Factor-Based Strategies to Generate Neural Cell Types from Human Pluripotent Stem Cells.

In the last years, the use of pluripotent stem cells in studies of human biology has grown exponentially. These cells represent an infinite source for differentiation into several human cell types facilitating the investigation on biological processes, functionality of cells, or diseases mechanisms in relevant human models. In the neurobiology field, pluripotent stem cells have been extensively used to generate the main neuronal and glial cells of the brain. Traditionally, protocols following developmental cues have been applied to pluripotent stem cells to drive differentiation toward different cell lineages; however, these protocols give rise to populations with mixed identities. Interestingly, new protocols applying overexpression of lineage-specific transcription factors (TFs) have emerged and facilitated the generation of highly pure populations of specific subtypes of neurons and glial cells in an easy, reproducible, and rapid manner. In this study, we review protocols based on this strategy to generate excitatory, inhibitory, dopaminergic, and motor neurons as well as astrocytes, oligodendrocytes, and microglia. In addition, we will discuss the main applications for cells generated with these protocols, including disease modeling, drug screening, and mechanistic studies. Finally, we will discuss the advantages and disadvantages of TF-based protocols and present our view of the future in this field.

Transcription Factor Programming of Human Pluripotent Stem Cells to Functionally Mature Astrocytes for Monocultures and Cocultures with Neurons.

Astrocytes are essential cells for normal brain functionality and have recently emerged as key players in many neurological diseases. However, the limited availability of human primary astrocytes for cell culture studies hinders our understanding of their physiology and precise role in disease development and progression. Here, we describe a detailed step-by-step protocol to rapidly and efficiently generate functionally mature induced astrocytes (iAs) from human embryonic and induced pluripotent stem cells (hES/iPSCs). Astrocyte induction is accomplished by ectopic lentiviral expression of two gliogenic transcription factors, Sox9 and Nfib. iAs exhibit morphology features as well as gene and protein expression similar to human mature astrocytes and display important astrocytic functions, such as glutamate uptake, propagation of calcium waves, expression of various cytokines after stimulation, and support of synapse formation and function, making them suitable models for studying the role of astrocytes in health and disease. Moreover, we describe a procedure for cryopreservation of iAs for long-term storage or shipping. Finally, we provide the required information needed to set up cocultures with human induced neurons (iNs, also described in this book), generated from hES/iPSCs, to generate cocultures, allowing studies on astrocyte-neuron interactions and providing new insights in astrocyte-associated disease mechanisms.

CRISPR/Cas9 Genome Engineering in Human Pluripotent Stem Cells for Modeling of Neurological Disorders.

Recent advances in genome editing have brought new hopes for personalized and precision medicine but have also dramatically facilitated disease modeling studies. Combined with reprogramming approaches, stem cells and differentiation toward neural lineages, genome engineering holds great potential for regenerative approaches and to model neurological disorders. The use of patient-specific induced pluripotent stem cells combined with neural differentiation allows studying the effect of specific mutations in different brain cells. New genome editing tools such as CRISPR/Cas9 represent a step further by facilitating the introduction or correction of specific mutations within the same cell line, thus eliminating variability due to differences in the genetic background. Here, we present a step-by-step protocol from design to generation of human pluripotent stem cell lines with specific mutations introduced or corrected with CRISPR/Cas9 gene editing that can be used in combination with transcription factor-based protocols to dissect underlying mechanisms of neurological disorders.

Loss of Cxcr5 alters neuroblast proliferation and migration in the aged brain.

Neurogenesis, the production of new neurons from neural stem cells, dramatically decreases during aging concomitantly with increased inflammation both systemically and in the brain. However, the precise role of inflammation and whether local or systemic factors drive the neurogenic decline during aging is poorly understood. Here, we identify CXCR5/5/CXCL13 signaling as a novel regulator of neurogenesis in the aged brain. The chemokine Cxcl13 was found to be upregulated in the brain during aging. Loss of its receptor, Cxcr5, led to increased proliferation and decreased numbers of neuroblasts in the aged subventricular zone (SVZ), together with accumulation of neuroblasts in the rostral migratory stream and olfactory bulb (OB), without increasing the amount of new mature neurons in the OB. The effect on proliferation and migration was specific to neuroblasts and likely mediated through increased levels of systemic IL-6 and local Cxcl12 expression in the SVZ. Our study raises the possibility of a new mechanism by which interplay between systemic and local alterations in inflammation regulates neurogenesis during aging.

Mitochondrial Dysfunction and Calcium Dysregulation in Leigh Syndrome Induced Pluripotent Stem Cell Derived Neurons.

Leigh syndrome (LS) is the most frequent infantile mitochondrial disorder (MD) and is characterized by neurodegeneration and astrogliosis in the basal ganglia or the brain stem. At present, there is no cure or treatment for this disease, partly due to scarcity of LS models. Current models generally fail to recapitulate important traits of the disease. Therefore, there is an urgent need to develop new human in vitro models. Establishment of induced pluripotent stem cells (iPSCs) followed by differentiation into neurons is a powerful tool to obtain an in vitro model for LS. Here, we describe the generation and characterization of iPSCs, neural stem cells (NSCs) and iPSC-derived neurons harboring the mtDNA mutation m.13513G>A in heteroplasmy. We have performed mitochondrial characterization, analysis of electrophysiological properties and calcium imaging of LS neurons. Here, we show a clearly compromised oxidative phosphorylation (OXPHOS) function in LS patient neurons. This is also the first report of electrophysiological studies performed on iPSC-derived neurons harboring an mtDNA mutation, which revealed that, in spite of having identical electrical properties, diseased neurons manifested mitochondrial dysfunction together with a diminished calcium buffering capacity. This could lead to an overload of cytoplasmic calcium concentration and the consequent cell death observed in patients. Importantly, our results highlight the importance of calcium homeostasis in LS pathology.

Neuronal and Astrocytic Differentiation from Sanfilippo C Syndrome iPSCs for Disease Modeling and Drug Development.

Sanfilippo syndrome type C (mucopolysaccharidosis IIIC) is an early-onset neurodegenerative lysosomal storage disorder, which is currently untreatable. The vast majority of studies focusing on disease mechanisms of Sanfilippo syndrome were performed on non-neural cells or mouse models, which present obvious limitations. Induced pluripotent stem cells (iPSCs) are an efficient way to model human diseases in vitro. Recently developed transcription factor-based differentiation protocols allow fast and efficient conversion of iPSCs into the cell type of interest. By applying these protocols, we have generated new neuronal and astrocytic models of Sanfilippo syndrome using our previously established disease iPSC lines. Moreover, our neuronal model exhibits disease-specific molecular phenotypes, such as increase in lysosomes and heparan sulfate. Lastly, we tested an experimental, siRNA-based treatment previously shown to be successful in patients' fibroblasts and demonstrated its lack of efficacy in neurons. Our findings highlight the need to use relevant human cellular models to test therapeutic interventions and shows the applicability of our neuronal and astrocytic models of Sanfilippo syndrome for future studies on disease mechanisms and drug development.

In Vitro Functional Characterization of Human Neurons and Astrocytes Using Calcium Imaging and Electrophysiology.

Recent progress in stem cell biology and epigenetic reprogramming has opened up previously unimaginable possibilities to study and develop regenerative approaches for neurological disorders. Human neurons and glial cells can be generated by differentiation of embryonic and neural stem cells and from somatic cells through reprogramming to pluripotency (followed by differentiation) as well as by direct conversion. All of these cells have the potential to be used for studying and treating neurological disorders. However, before considering using human neural cells derived from these sources for modelling or regenerative purposes, they need to be verified in terms of functionality and similarity to endogenous cells in the central nervous system (CNS).In this chapter, we describe how to assess functionality of neurons and astrocytes derived from stem cells and through direct reprogramming, using calcium imaging and electrophysiology.

Author Correction: Rapid and efficient induction of functional astrocytes from human pluripotent stem cells.

In the version of Supplementary Fig. 1 originally published with this paper, some images in panel e were accidental duplicates of images in panel b. This error has been corrected in the online integrated supplementary information and in the Supplementary Information PDF.

Transcription factor programming of human ES cells generates functional neurons expressing both upper and deep layer cortical markers.

Human neurodegenerative disorders affect specific types of cortical neurons. Efficient protocols for the generation of such neurons for cell replacement, disease modeling and drug screening are highly warranted. Current methods for the production of cortical neurons from human embryonic stem (ES) cells are often time-consuming and inefficient, and the functional properties of the generated cells have been incompletely characterized. Here we have used transcription factor (TF) programming with the aim to induce rapid differentiation of human ES cells to layer-specific cortical neurons (hES-iNs). Three different combinations of TFs, NEUROGENIN 2 (NGN2) only, NGN2 plus Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2), and NGN2 plus Special AT-Rich Sequence-Binding Protein 2 (SATB2), were delivered to human ES cells by lentiviral vectors. We observed only subtle differences between the TF combinations, which all gave rise to the formation of pyramidal-shaped cells, morphologically resembling adult human cortical neurons expressing cortical projection neuron (PN) markers and with mature electrophysiological properties. Using ex vivo transplantation to human organotypic cultures, we found that the hES-iNs could integrate into adult human cortical networks. We obtained no evidence that the hES-iNs had acquired a distinct cortical layer phenotype. Instead, our single-cell data showed that the hES-iNs, similar to fetal human cortical neurons, expressed both upper and deep layer cortical neuronal markers. Taken together, our findings provide evidence that TF programming can direct human ES cells towards cortical neurons but that the generated cells are transcriptionally profiled to generate both upper and deep layer cortical neurons. Therefore, most likely additional cues will be needed if these cells should adopt a specific cortical layer and area identity.

Groucho related gene 5 (GRG5) is involved in embryonic and neural stem cell state decisions.

Groucho related gene 5 (GRG5) is a multifunctional protein that has been implicated in late embryonic and postnatal mouse development. Here, we describe a previously unknown role of GRG5 in early developmental stages by analyzing its function in stem cell fate decisions. By both loss and gain of function approaches we demonstrate that ablation of GRG5 deregulates the Embryonic Stem Cell (ESC) pluripotent state whereas its overexpression leads to enhanced self-renewal and acquisition of cancer cell-like properties. The malignant characteristics of teratomas generated by ESCs that overexpress GRG5 reveal its pro-oncogenic potential. Furthermore, transcriptomic analysis and cell differentiation approaches underline GRG5 as a multifaceted signaling regulator that represses mesendodermal-related genes. When ESCs exit pluripotency, GRG5 promotes neuroectodermal specification via Wnt and BMP signaling suppression. Moreover, GRG5 promotes the neuronal reprogramming of fibroblasts and maintains the self-renewal of Neural Stem Cells (NSCs) by sustaining the activity of Notch/Hes and Stat3 signaling pathways. In summary, our results demonstrate that GRG5 has pleiotropic roles in stem cell biology functioning as a stemness factor and a neural fate specifier.

Rapid and efficient induction of functional astrocytes from human pluripotent stem cells.

The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease.

Direct conversion of human fibroblasts to functional excitatory cortical neurons integrating into human neural networks.

BACKGROUND: Human fibroblasts can be directly converted to several subtypes of neurons, but cortical projection neurons have not been generated. METHODS: Here we screened for transcription factor combinations that could potentially convert human fibroblasts to functional excitatory cortical neurons. The induced cortical (iCtx) cells were analyzed for cortical neuronal identity using immunocytochemistry, single-cell quantitative polymerase chain reaction (qPCR), electrophysiology, and their ability to integrate into human neural networks in vitro and ex vivo using electrophysiology and rabies virus tracing. RESULTS: We show that a combination of three transcription factors, BRN2, MYT1L, and FEZF2, have the ability to directly convert human fibroblasts to functional excitatory cortical neurons. The conversion efficiency was increased to about 16% by treatment with small molecules and microRNAs. The iCtx cells exhibited electrophysiological properties of functional neurons, had pyramidal-like cell morphology, and expressed key cortical projection neuronal markers. Single-cell analysis of iCtx cells revealed a complex gene expression profile, a subpopulation of them displaying a molecular signature closely resembling that of human fetal primary cortical neurons. The iCtx cells received synaptic inputs from co-cultured human fetal primary cortical neurons, contained spines, and expressed the postsynaptic excitatory scaffold protein PSD95. When transplanted ex vivo to organotypic cultures of adult human cerebral cortex, the iCtx cells exhibited morphological and electrophysiological properties of mature neurons, integrated structurally into the cortical tissue, and received synaptic inputs from adult human neurons. CONCLUSIONS: Our findings indicate that functional excitatory cortical neurons, generated here for the first time by direct conversion of human somatic cells, have the capacity for synaptic integration into adult human cortex.

Generation of pure GABAergic neurons by transcription factor programming.

Approaches to differentiating pluripotent stem cells (PSCs) into neurons currently face two major challenges-(i) generated cells are immature, with limited functional properties; and (ii) cultures exhibit heterogeneous neuronal subtypes and maturation stages. Using lineage-determining transcription factors, we previously developed a single-step method to generate glutamatergic neurons from human PSCs. Here, we show that transient expression of the transcription factors Ascl1 and Dlx2 (AD) induces the generation of exclusively GABAergic neurons from human PSCs with a high degree of synaptic maturation. These AD-induced neuronal (iN) cells represent largely nonoverlapping populations of GABAergic neurons that express various subtype-specific markers. We further used AD-iN cells to establish that human collybistin, the loss of gene function of which causes severe encephalopathy, is required for inhibitory synaptic function. The generation of defined populations of functionally mature human GABAergic neurons represents an important step toward enabling the study of diseases affecting inhibitory synaptic transmission.