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Ras/MAPK pathway signaling is a major participant in neurodevelopment, and evidence suggests that BRAF, a key Ras signal mediator, influences human behavior. We studied the role of the mutation BRAFQ257R, the most common cause of cardiofaciocutaneous syndrome (CFC), in an induced pluripotent stem cell (iPSC)-derived model of human neurodevelopment. In iPSC-derived neuronal cultures from CFC subjects, we observed decreased p-AKT and p-ERK1/2 compared to controls, as well as a depleted neural progenitor pool and rapid neuronal maturation. Pharmacological PI3K/AKT pathway manipulation recapitulated cellular phenotypes in control cells and attenuated them in CFC cells. CFC cultures displayed altered cellular subtype ratios and increased intrinsic excitability. Moreover, in CFC cells, Ras/MAPK pathway activation and morphological abnormalities exhibited cell subtype-specific differences. Our results highlight the importance of exploring specific cellular subtypes and of using iPSC models to reveal relevant human-specific neurodevelopmental events.

What roles do astrocytes play in human disease? This question remains unanswered for nearly every human neurological disorder. Yet, because of their abundance and complexity astrocytes can impact neurological function in many ways. The differentiation of h

Biological neural networks exhibit synchronized activity within and across interconnected regions of the central nervous system. Understanding how these coordinated networks are established and maintained may reveal therapeutic targets for neurodegeneration and neuromodulation. Here, we tested the influence of astrocytes upon synchronous network activity using human pluripotent stem cell-derived bioengineered neural organoids. This study revealed that astrocytes significantly increase activity within individual organoids and across long distances among numerous rapidly merged organoids via influencing synapses and bioenergetics. Treatment of amyloid protein inhibited synchronous activity during neurodegeneration, yet this can be rescued by propagating activity from neighboring networks. Altogether, this study identifies critical contributions of human astrocytes to biological neural networks and delivers a rapid, reproducible, and scalable model to investigate long-range functional communication of the nervous system in healthy and disease states.

Using machine learning (ML), we interrogated the function of all human-chimpanzee variants in 2,645 human accelerated regions (HARs), finding 43% of HARs have variants with large opposing effects on chromatin state and 14% on neurodevelopmental enhancer activity. This pattern, consistent with compensatory evolution, was confirmed using massively parallel reporter assays in chimpanzee and human neural progenitor cells. The species-specific enhancer activity of HARs was accurately predicted from the presence and absence of transcription factor footprints in each species. Despite these striking cis effects, activity of a given HAR sequence was nearly identical in human and chimpanzee cells. This suggests that HARs did not evolve to compensate for changes in the trans environment but instead altered their ability to bind factors present in both species. Thus, ML prioritized variants with functional effects on human neurodevelopment and revealed an unexpected reason why HARs may have evolved so rapidly., Competing Interests: Declaration of interests A.K. is a cofounder, consultant, and member of the board of Neurona Therapeutic, a company studying the potential therapeutic use of interneuron transplantation. J.L.R. is a cofounder, stockholder, and member of the board of Neurona. N.A. is a cofounder and on the scientific advisory board of Regel Therapeutics and Neomer Diagnostics and receives funding from BioMarin Pharmaceutical Incorporate. K.K. is currently an employee of Fauna Bio., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Astrocyte reactivity can directly modulate nervous system function and immune responses during disease and injury. However, the consequence of human astrocyte reactivity in response to specific contexts and within neural networks is obscure. Here, we devised a straightforward bioengineered neural organoid culture approach entailing transcription factor-driven direct differentiation of neurons and astrocytes from human pluripotent stem cells combined with genetically encoded tools for dual cell-selective activation. This strategy revealed that Gq-GPCR activation via chemogenetics in astrocytes promotes a rise in intracellular calcium followed by induction of immediate early genes and thrombospondin 1. However, astrocytes also undergo NF-κB nuclear translocation and secretion of inflammatory proteins, correlating with a decreased evoked firing rate of cocultured optogenetic neurons in suboptimal conditions, without overt neurotoxicity. Altogether, this study clarifies the intrinsic reactivity of human astrocytes in response to targeting GPCRs and delivers a bioengineered approach for organoid-based disease modeling and preclinical drug testing., (© 2022 Cvetkovic et al.)

Host immune cells interact bi-directionally with their extracellular matrix (ECM) to receive and deposit molecular signals, which orchestrate cellular activation, proliferation, differentiation, and function to maintain healthy tissue homeostasis. In response to pathogens or damage, immune cells infiltrate diseased sites and synthesize critical ECM molecules such as glycoproteins, proteoglycans, and glycosaminoglycans to promote healing. When the immune system misidentifies pathogens or fails to survey damaged cells effectively, maladies such as chronic inflammation, autoimmune diseases, and cancer can develop. In these conditions, it is essential to restore balance to the body through modulation of the immune system and the ECM. This review details the components of dysregulated ECM implicated in pathogenic environments and therapeutic approaches to restore tissue homeostasis. We evaluate emerging strategies to overcome inflamed, immune inhibitory, and otherwise diseased microenvironments, including mechanical stimulation, targeted proteases, adoptive cell therapy, mechanomedicine, and biomaterial-based cell therapeutics. We highlight various strategies that have produced efficacious responses in both pre-clinical and human trials and identify additional opportunities to develop next-generation interventions. Significantly, we identify a need for therapies to address dense or fibrotic tissue for the treatment of organ tissue damage and various cancer subtypes. Finally, we conclude that therapeutic techniques that disrupt, evade, or specifically target the pathogenic microenvironment have a high potential for improving therapeutic outcomes and should be considered a priority for immediate exploration. A schematic showing the various methods of extracellular matrix disruption/targeting in both fibrotic and cancerous environments. a Biomaterial-based cell therapy can be used to deliver anti-inflammatory cytokines, chemotherapeutics, or other factors for localized, slow release of therapeutics. b Mechanotherapeutics can be used to inhibit the deposition of molecules such as collagen that affect stiffness. c Ablation of the ECM and target tissue can be accomplished via mechanical degradation such as focused ultrasound. d Proteases can be used to improve the distribution of therapies such as oncolytic virus. e Localization of therapeutics such as checkpoint inhibitors can be improved with the targeting of specific ECM components, reducing off-target effects and toxicity., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Intracerebral hemorrhage (ICH) remains a common and debilitating form of stroke. This neurological emergency must be diagnosed and treated rapidly yet effectively. In this article, we review the medical, surgical, repair, and regenerative treatment options for managing ICH. Topics of focus include the management of blood pressure, intracranial pressure, coagulopathy, and intraventricular hemorrhage, as well as the role of surgery, regeneration, rehabilitation, and secondary prevention. Results of various phase II and III trials are incorporated. In summary, ICH patients should undergo rapid evaluation with neuroimaging, and early interventions should include systolic blood pressure control in the range of 140 mmHg, correction of coagulopathy if indicated, and assessment for surgical intervention. ICH patients should be managed in dedicated neurosurgical intensive care or stroke units where continuous monitoring of neurological status and evaluation for neurological deterioration is rapidly possible. Extravasation of hematoma may be helpful in patients with intraventricular extension of ICH. The goal of care is to reduce mortality and enable multimodal rehabilitative therapy., (© 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Nanovesicles (NVs) are emerging as innovative, theranostic tools for cargo delivery. Recently, surface engineering of NVs with membrane proteins from specific cell types has been shown to improve the biocompatibility of NVs and enable the integration of functional attributes. However, this type of biomimetic approach has not yet been explored using human neural cells for applications within the nervous system. Here, this paper optimizes and validates the scalable and reproducible production of two types of neuron-targeting NVs, each with a distinct lipid formulation backbone suited to potential therapeutic cargo, by integrating membrane proteins that are unbiasedly sourced from human pluripotent stem-cell-derived neurons. The results establish that both endogenous and genetically engineered cell-derived proteins effectively transfer to NVs without disruption of their physicochemical properties. NVs with neuron-derived membrane proteins exhibit enhanced neuronal association and uptake compared to bare NVs. Viability of 3D neural sphere cultures is not disrupted by treatment, which verifies the utility of organoid-based approaches as NV testing platforms. Finally, these results confirm cellular association and uptake of the biomimetic humanized NVs to neurons within rodent cranial nerves. In summary, the customizable NVs reported here enable next-generation functionalized theranostics aimed to promote neuroregeneration., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

CAG repeat expansion is the genetic cause of nine incurable polyglutamine (polyQ) diseases with neurodegenerative features. Silencing repeat RNA holds great therapeutic value. Here, we developed a repeat-based RNA-cleaving DNAzyme that catalyzes the destruction of expanded CAG repeat RNA of six polyQ diseases with high potency. DNAzyme preferentially cleaved the expanded allele in spinocerebellar ataxia type 1 (SCA1) cells. While cleavage was non-allele-specific for spinocerebellar ataxia type 3 (SCA3) cells, treatment of DNAzyme leads to improved cell viability without affecting mitochondrial metabolism or p62-dependent aggresome formation. DNAzyme appears to be stable in mouse brain for at least 1 month, and an intermediate dosage of DNAzyme in a SCA3 mouse model leads to a significant reduction of high molecular weight ATXN3 proteins. Our data suggest that DNAzyme is an effective RNA silencing molecule for potential treatment of multiple polyQ diseases., (© 2021. The Author(s).)

Astrocytes play central roles in normal brain function and are critical components of synaptic networks that oversee behavioral outputs. Despite their close affiliation with neurons, how neuronal-derived signals influence astrocyte function at the gene expression level remains poorly characterized, largely due to difficulties associated with dissecting neuron- versus astrocyte-specific effects. Here, we use an in vitro system of stem cell-derived astrocytes to identify gene expression profiles in astrocytes that are influenced by neurons and regulate astrocyte development. Furthermore, we show that neurotransmitters and neuromodulators induce distinct transcriptomic and chromatin accessibility changes in astrocytes that are unique to each of these neuroactive compounds. These findings are highlighted by the observation that noradrenaline has a more profound effect on transcriptional profiles of astrocytes compared to glutamate, gamma-aminobutyric acid (GABA), acetylcholine, and serotonin. This is demonstrated through enhanced noradrenaline-induced transcriptomic and chromatin accessibility changes in vitro and through enhanced calcium signaling in vivo. Taken together, our study reveals distinct transcriptomic and chromatin architecture signatures in astrocytes in response to neuronal-derived neuroactive compounds. Since astrocyte function is affected in all neurological disorders, this study provides a new entry point for exploring genetic mechanisms of astrocyte-neuron communication that may be dysregulated in disease.

Astrocytes exhibit dynamic and complex reactions to various insults. Recently, investigations into the transitions that occur during cellular specification, differentiation, maturation, and disease responses have provided insights into understanding the mechanisms that underlie these altered states of reactivity and function. Here we summarize current concepts in how astrocyte state transitions, termed astroplasticity, are regulated, as well as how this affects neural circuit function through extracellular signaling. We postulate that a promising future approach toward enhancing functional repair after injury and disease would be to steer astrocytes away from an inhibitory response and toward one that is beneficial to neuroplasticity and neuroregeneration. Toward this goal, we discuss emerging biotechnological advancements, with a focus on human pluripotent stem cell bioengineering, which has high potential for effective manipulation and control of astroplasticity. Highlights include innovations in cellular transdifferentiation techniques, nanomedicine, organoid and three-dimensional (3D) spheroid microcircuit development, and the use of biomaterials to influence the extracellular environment. Current barriers and future applications are also summarized in order to augment the design of future preclinical trials aimed toward astrocyte-targeted neuroregeneration with a concept termed astrocellular therapeutics. Developmental Dynamics 248:21-33, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

How mutations in glial fibrillary acidic protein (GFAP) cause Alexander disease (AxD) remains elusive. We generated iPSCs from two AxD patients and corrected the GFAP mutations to examine the effects of mutant GFAP on human astrocytes. AxD astrocytes displayed GFAP aggregates, recapitulating the pathological hallmark of AxD. RNA sequencing implicated the endoplasmic reticulum, vesicle regulation, and cellular metabolism. Corroborating this analysis, we observed enlarged and heterogeneous morphology coupled with perinuclear localization of endoplasmic reticulum and lysosomes in AxD astrocytes. Functionally, AxD astrocytes showed impaired extracellular ATP release, which is responsible for attenuated calcium wave propagation. These results reveal that AxD-causing mutations in GFAP disrupt intracellular vesicle regulation and impair astrocyte secretion, resulting in astrocyte dysfunction and AxD pathogenesis., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed 'organoids' or 'spheroids', for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures ('asteroids') are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

Human astrocytes network with neurons in dynamic ways that are still poorly defined. Our ability to model this relationship is hampered by the lack of relevant and convenient tools to recapitulate this complex interaction. To address this barrier, we have devised efficient coculture systems utilizing 3D organoid-like spheres, termed asteroids, containing pre-differentiated human pluripotent stem cell (hPSC)-derived astrocytes (hAstros) combined with neurons generated from hPSC-derived neural stem cells (hNeurons) or directly induced via Neurogenin 2 overexpression (iNeurons). Our systematic methods rapidly produce structurally complex hAstros and synapses in high-density coculture with iNeurons in precise numbers, allowing for improved studies of neural circuit function, disease modeling, and drug screening. We conclude that these bioengineered neural circuit model systems are reliable and scalable tools to accurately study aspects of human astrocyte-neuron functional properties while being easily accessible for cell-type-specific manipulations and observations., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype-dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery., (© 2017 Krejciova et al.)

Cellular components of synaptic circuits have been adjusted for increased human brain size, neural cell density, energy consumption and developmental duration. How does the human brain make these accommodations? There is evidence that astrocytes are one of the most divergent neural cell types in primate brain evolution and it is now becoming clear that they have critical roles in controlling synaptic development, function and plasticity. Yet, we still do not know how the precise developmental appearance of these cells and subsequent astrocyte-derived signals modulate diverse neuronal circuit subtypes. Here, we discuss what is currently known about the influence of glial factors on synaptic maturation and focus on unique features of human astrocytes including their potential roles in regenerative and translational medicine. Human astrocyte distinctiveness may be a major contributor to high level neuronal processing of the human brain and act in novel ways during various neuropathies ranging from autism spectrum disorders, viral infection, injury and neurodegenerative conditions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic., Competing Interests: The authors declare no conflict of interest.

Background: Basal forebrain cholinergic neurons (BFCNs) play critical roles in learning, memory and cognition. Dysfunction or degeneration of BFCNs may connect to neuropathology, such as Alzheimer's disease, Down's syndrome and dementia. Generation of functional BFCNs may contribute to the studies of cell-based therapy and pathogenesis that is related to learning and memory deficits., New Method: Here we describe a detail method for robust generation of BFCNs from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). In this method, BFCN progenitors are patterned from hESC or hiPSC-derived primitive neuroepithelial cells, with the treatment of sonic hedgehog (SHH) or combination with its agonist Purmorphamine, and by co-culturing with human astrocytes., Results: At day 20, ∼90% hPSC-derived progenitors expressed NKX2.1, which is a transcriptional marker for MGE. Moreover, around 40% of NKX2.1+ cells co-expressed OLIG2 and ∼15% of NKX2.1+ cells co-expressed ISLET1, which are ventral markers. At day 35, ∼40% neurons robustly express ChAT, most of which are co-labeled with NKX2.1, ISLET1 and FOXG1, indicating the basal forebrain-like identity. At day 45, these neurons express mature neuronal markers MAP2, Synapsin, and VAChT., Comparison With Existing Method(s): In this method, undefined conditions including genetic modification or cell-sorting are avoided. As a choice, feeder free conditions are used to avoid ingredients of animal origin. Moreover, Purmorphamine can be substituted for SHH to induce ventral progenitors effectively and economically., Conclusion: We provide an efficient method to generate BFCNs from multiple hPSC lines, which offers the potential application for disease modeling and pharmacological studies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Human pluripotent stem cells (hPSCs) have potential to differentiate to unlimited number of neural cells, which provide powerful tools for neural regeneration. To date, most reported protocols were established with an animal feeder system. However, cells derived on this system are inappropriate for the translation to clinical applications because of the introduction of xenogenetic factors. In this study, we provided an optimized paradigm to generate region-specific forebrain neurons from hPSCs under a defined system. We assessed five conditions and found that a vitronectin-coated substrate was the most efficient method to differentiate hPSCs to neurons and astrocytes. More importantly, by applying different doses of purmorphamine, a small-molecule agonist of sonic hedgehog signaling, hPSCs were differentiated to different region-specific forebrain neuron subtypes, including glutamatergic neurons, striatal medium spiny neurons, and GABA interneurons. Our study offers a highly defined system without exogenetic factors to produce human neurons and astrocytes for translational medical studies, including cell therapy and stem cell-based drug discovery.

Astrocytes produce an assortment of signals that promote neuronal maturation according to a precise developmental timeline. Is this orchestrated timing and signaling altered in human neurodevelopmental disorders? To address this question, the astroglial lineage was investigated in two model systems of a developmental disorder with intellectual disability caused by mutant Harvey rat sarcoma viral oncogene homolog (HRAS) termed Costello syndrome: mutant HRAS human induced pluripotent stem cells (iPSCs) and transgenic mice. Human iPSCs derived from patients with Costello syndrome differentiated to astroglia more rapidly in vitro than those derived from wild-type cell lines with normal HRAS, exhibited hyperplasia, and also generated an abundance of extracellular matrix remodeling factors and proteoglycans. Acute treatment with a farnesyl transferase inhibitor and knockdown of the transcription factor SNAI2 reduced expression of several proteoglycans in Costello syndrome iPSC-derived astrocytes. Similarly, mice in which mutant HRAS was expressed selectively in astrocytes exhibited experience-independent increased accumulation of perineuronal net proteoglycans in cortex, as well as increased parvalbumin expression in interneurons, when compared to wild-type mice. Our data indicate that astrocytes expressing mutant HRAS dysregulate cortical maturation during development as shown by abnormal extracellular matrix remodeling and implicate excessive astrocyte-to-neuron signaling as a possible drug target for treating mental impairment and enhancing neuroplasticity., (Copyright © 2015, American Association for the Advancement of Science.)

Dysfunction of basal forebrain cholinergic neurons (BFCNs) and γ-aminobutyric acid (GABA) interneurons, derived from medial ganglionic eminence (MGE), is implicated in disorders of learning and memory. Here we present a method for differentiating human embryonic stem cells (hESCs) to a nearly uniform population of NKX2.1(+) MGE-like progenitor cells. After transplantation into the hippocampus of mice in which BFCNs and some GABA neurons in the medial septum had been destroyed by mu P75-saporin, human MGE-like progenitors, but not ventral spinal progenitors, produced BFCNs that synaptically connected with endogenous neurons, whereas both progenitors generated similar populations of GABA neurons. Mice transplanted with MGE-like but not spinal progenitors showed improvements in learning and memory deficits. These results suggest that progeny of the MGE-like progenitors, particularly BFCNs, contributed to learning and memory. Our findings support the prospect of using human stem cell-derived MGE-like progenitors in developing therapies for neurological disorders of learning and memory.

What roles do astrocytes play in human disease?This question remains unanswered for nearly every human neurological disorder. Yet, because of their abundance and complexity astrocytes can impact neurological function in many ways. The differentiation of human pluripotent stem cells (hPSCs) into neuronal and glial subtypes, including astrocytes, is becoming routine, thus their use as tools for modeling neurodevelopment and disease will provide one important approach to answer this question. When designing experiments, careful consideration must be given to choosing paradigms for differentiation, maturation, and functional analysis of these temporally asynchronous cellular populations in culture. In the case of astrocytes, they display heterogeneous characteristics depending upon species of origin, brain region, developmental stage, environmental factors, and disease states, all of which may render experimental results highly variable. In this review, challenges and future directions are discussed for using hPSC-derived astroglial progenitors and mature astrocytes for neurodevelopmental studies with a focus on exploring human astrocyte effects upon neuronal function. As new technologies emerge to measure the functions of astrocytes in vitro and in vivo, there is also a need for a standardized source of human astrocytes that are most relevant to the diseases of interest.

Astrocytes are no longer seen as a homogenous population of cells. In fact, recent studies indicate that astrocytes are morphologically and functionally diverse and play critical roles in neurodevelopmental diseases such as Rett syndrome and fragile X mental retardation. This review summarizes recent advances in astrocyte development, including the role of neural tube patterning in specification and developmental functions of astrocytes during synaptogenesis. We propose here that a precise understanding of astrocyte development is critical to defining heterogeneity and could lead advances in understanding and treating a variety of neuropsychiatric diseases.

Regionally and functionally diverse types of astrocytes exist throughout the central nervous system and participate in nearly every aspect of normal and abnormal neural function. Therefore, human astrocyte subtypes are useful tools for understanding brain function, modulating disease processes and promoting neural regeneration. Here we describe a protocol for directed differentiation and maintenance of functional astroglia from human pluripotent stem cells in a chemically defined system. Human stem cells are first differentiated into neuroepithelial cells with or without exogenous patterning molecules (days 0-21). Regular dissociation of the neuroepithelial clusters in suspension, and in the presence of mitogens, permits generation of astroglial subtypes over a long-term expansion (days 21-90). Finally, the astroglial progenitors are either amplified for an extended time or differentiated into functional astrocytes on removal of mitogens and the addition of ciliary neurotrophic factor (days >90). This method generates robust populations of functionally diversified astrocytes with high efficiency.

Human pluripotent stem cells (hPSCs) have been differentiated efficiently to neuronal cell types. However, directed differentiation of hPSCs to astrocytes and astroglial subtypes remains elusive. In this study, hPSCs were directed to nearly uniform populations of immature astrocytes (>90% S100β(+) and GFAP(+)) in large quantities. The immature human astrocytes exhibit similar gene expression patterns as primary astrocytes, display functional properties such as glutamate uptake and promotion of synaptogenesis, and become mature astrocytes by forming connections with blood vessels after transplantation into the mouse brain. Furthermore, hPSC-derived neuroepithelia, patterned to rostral-caudal and dorsal-ventral identities with the same morphogens used for neuronal subtype specification, generate immature astrocytes that express distinct homeodomain transcription factors and display phenotypic differences of different astroglial subtypes. These human astroglial progenitors and immature astrocytes will be useful for studying astrocytes in brain development and function, understanding the roles of astrocytes in disease processes and developing novel treatments for neurological disorders.

The JIL-1 histone H3S10 kinase in Drosophila localizes specifically to euchromatic interband regions of polytene chromosomes and is enriched 2-fold on the male X chromosome. JIL-1 can be divided into four main domains including an NH(2)-terminal domain, two separate kinase domains, and a COOH-terminal domain. Our results demonstrate that the COOH-terminal domain of JIL-1 is necessary and sufficient for correct chromosome targeting to autosomes but that both COOH- and NH(2)-terminal sequences are necessary for enrichment on the male X chromosome. We furthermore show that a small 53-amino acid region within the COOH-terminal domain can interact with the tail region of histone H3, suggesting that this interaction is necessary for the correct chromatin targeting of the JIL-1 kinase. Interestingly, our data indicate that the COOH-terminal domain alone is sufficient to rescue JIL-1 null mutant polytene chromosome defects including those of the male X chromosome. Nonetheless, we also found that a truncated JIL-1 protein which was without the COOH-terminal domain but retained histone H3S10 kinase activity was able to rescue autosome as well as partially rescue male X polytene chromosome morphology. Taken together these findings indicate that JIL-1 may participate in regulating chromatin structure by multiple and partially redundant mechanisms.

We have used a yeast two-hybrid screen to identify lamin Dm0 as an interaction partner for the nuclear JIL-1 kinase. This molecular interaction was confirmed by GST-fusion protein pull-down assays and by co-immunoprecipitation experiments. Using deletion construct analysis we show that a predicted globular domain of the basic region of the COOH-terminal domain of JIL-1 was sufficient for mediating the molecular interactions with lamin Dm0. A reciprocal analysis with truncated lamin Dm0 constructs showed that the interaction with JIL-1 required sequences in the tail domain of lamin Dm0 that include the Ig-like fold. Further support for a molecular interaction between JIL-1 and lamin Dm0 in vivo was provided by genetic interaction assays. We show that nuclear positioning and lamina morphology were abnormal in JIL-1 mutant egg chambers. The most common phenotypes observed were abnormal nurse cell nuclear lamina protrusions through the ring canals near the oocyte, as well as dispersed and mislocalized lamin throughout the egg chamber. These phenotypes were completely rescued by a full-length JIL-1 transgenic construct. Thus, our results suggest that the JIL-1 kinase is required to maintain nuclear morphology and integrity of nurse cells during oogenesis and that this function may be linked to molecular interactions with lamin Dm0.

Chronic mitochondrial dysfunction, in particular of complex I, has been strongly implicated in the dopaminergic neurodegeneration in Parkinson's disease. To elucidate the mechanisms of chronic complex I disruption-induced neurodegeneration, we induced differentiation of immortalized midbrain dopaminergic (MN9D) and non-dopaminergic (MN9X) neuronal cells, to maintain them in culture without significant cell proliferation and compared their survivals following chronic exposure to nanomolar rotenone, an irreversible complex I inhibitor. Rotenone killed more dopaminergic MN9D cells than non-dopaminergic MN9X cells. Oxidative stress played an important role in rotenone-induced neurodegeneration of MN9X cells, but not MN9D cells: rotenone oxidatively modified proteins more in MN9X cells than in MN9D cells and antioxidants decreased rotenone toxicity only in MN9X cells. MN9X cells were also more sensitive to exogenous oxidants than MN9D cells. In contrast, disruption of bioenergetics played a more important role in MN9D cells: rotenone decreased mitochondrial membrane protential and ATP levels in MN9D cells more than in MN9X cells. Supplementation of cellular energy with a ketone body, D-beta-hydroxybutyrate, decreased rotenone toxicity in MN9D cells, but not in MN9X cells. MN9D cells were also more susceptible to disruption of oxidative phosphorylation or glycolysis than MN9X cells. These findings indicate that, during chronic rotenone exposure, MN9D cells die primarily through mitochondrial energy disruption, whereas MN9X cells die primarily via oxidative stress. Thus, intrinsic properties of individual cell types play important roles in determining the predominant mechanism of complex I inhibition-induced neurodegeneration.