Patient-derived organoids recapitulate glioma-intrinsic immune program and progenitor populations of glioblastoma.

Glioblastoma multiforme (GBM) is a highly lethal human cancer thought to originate from a self-renewing and therapeutically-resistant population of glioblastoma stem cells (GSCs). The intrinsic mechanisms enacted by GSCs during 3D tumor formation, however, remain unclear, especially in the stages prior to angiogenic/immunological infiltration. In this study, we performed a deep characterization of the genetic, immune, and metabolic profiles of GBM organoids from several patient-derived GSCs (GBMO). Despite being devoid of immune cells, transcriptomic analysis across GBMO revealed a surprising immune-like molecular program, enriched in cytokine, antigen presentation and processing, T-cell receptor inhibitors, and interferon genes. We find two important cell populations thought to drive GBM progression, Special AT-rich sequence-binding protein 2 (SATB2+) and homeodomain-only protein homeobox (HOPX+) progenitors, contribute to this immune landscape in GBMO and GBM in vivo. These progenitors, but not other cell types in GBMO, are resistant to conventional GBM therapies, temozolomide and irradiation. Our work defines a novel intrinsic immune-like landscape in GBMO driven, in part, by SATB2+ and HOPX+ progenitors and deepens our understanding of the intrinsic mechanisms utilized by GSCs in early GBM formation.

Stat1 is an inducible transcriptional repressor of neural stem cells self-renewal program during neuroinflammation.

A central issue in regenerative medicine is understanding the mechanisms that regulate the self-renewal of endogenous stem cells in response to injury and disease. Interferons increase hematopoietic stem cells during infection by activating STAT1, but the mechanisms by which STAT1 regulates intrinsic programs in neural stem cells (NSCs) during neuroinflammation is less known. Here we explored the role of STAT1 on NSC self-renewal. We show that overexpressing Stat1 in NSCs derived from the subventricular zone (SVZ) decreases NSC self-renewal capacity while Stat1 deletion increases NSC self-renewal, neurogenesis, and oligodendrogenesis in isolated NSCs. Importantly, we find upregulation of STAT1 in NSCs in a mouse model of multiple sclerosis (MS) and an increase in pathological T cells expressing IFN-γ rather than interleukin 17 (IL-17) in the cerebrospinal fluid of affected mice. We find IFN-γ is superior to IL-17 in reducing proliferation and precipitating an abnormal NSC phenotype featuring increased STAT1 phosphorylation and Stat1 and p16ink4a gene expression. Notably, Stat1-/- NSCs were resistant to the effect of IFN-γ. Lastly, we identified a Stat1-dependent gene expression profile associated with an increase in the Sox9 transcription factor, a regulator of self-renewal. Stat1 binds and transcriptionally represses Sox9 in a transcriptional luciferase assay. We conclude that Stat1 serves as an inducible checkpoint for NSC self-renewal that is upregulated during chronic brain inflammation leading to decreased self-renewal. As such, Stat1 may be a potential target to modulate for next generation therapies to prevent progression and loss of repair function in NSCs/neural progenitors in MS.

SARS-CoV2 entry factors are expressed in primary human glioblastoma and recapitulated in cerebral organoid models.

PURPOSE: Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults with a median overall survival of only 14.6 months despite aggressive treatment. While immunotherapy has been successful in other cancers, its benefit has been proven elusive in GBM, mainly due to a markedly immunosuppressive tumor microenvironment. SARS-CoV-2 has been associated with the development of a pronounced central nervous system (CNS) inflammatory response when infecting different cells including astrocytes, endothelial cells, and microglia. While SARS-CoV2 entry factors have been described in different tissues, their presence and implication on GBM aggressiveness or microenvironment has not been studied on appropriate preclinical models. METHODS: We evaluated the presence of crucial SARS-CoV-2 entry factors: ACE2, TMPRSS2, and NRP1 in matched surgically-derived GBM tissue, cells lines, and organoids; as well as in human brain derived specimens using immunohistochemistry, confocal pixel line intensity quantification, and transcriptome analysis. RESULTS: We show that patient derived-GBM tissue and cell cultures express SARS-CoV2 entry factors, being NRP1 the most crucial facilitator of SARS-CoV-2 infection in GBM. Moreover, we demonstrate that, receptor expression remains present in our GBM organoids, making them an adequate model to study the effect of this virus in GBM for the potential development of viral therapies in the future. CONCLUSION: Our findings suggest that the SARS-CoV-2 virus entry factors are expressed in primary tissues and organoid models and could be potentially utilized to study the susceptibility of GBM to this virus to target or modulate the tumor microenviroment.

Development of Experimental Three-Dimensional Tumor Models to Study Glioblastoma Cancer Stem Cells and Tumor Microenvironment.

Glioblastoma (GBM) is the most common and dismal primary brain tumor. Unfortunately, despite multidisciplinary treatment, most patients will perish approximately 15 months after diagnosis. For this reason, there is an urgent need to improve our understanding of GBM tumor biology and develop novel therapies that can achieve better clinical outcomes. In this setting, three-dimensional tumor models have risen as more appropriate preclinical tools when compared to traditional cell cultures, given that two-dimensional (2D) cultures have failed to accurately recapitulate tumor biology and translate preclinical findings into patient benefits. Three-dimensional cultures using neurospheres, organoids, and organotypic better resemble original tumor genetic and epigenetic profiles, maintaining tumor microenvironment characteristics and mimicking cell-cell and cell-matrix interactions. This chapter summarizes our methods to generate well-characterized glioblastoma neurospheres, organoids, and organotypics.

Generation of Neurosphere-Derived Organoid-Like-Aggregates (NEDAS) from Neural Stem Cells.

Neurosphere cultures have been used to propagate and study the intrinsic properties of neural stem cells (NSCs) for more than two decades but this method has many limitations. It is well known that neurospheres fuse in culture, but the long-term biological consequences of this phenomena are not well characterized. We leveraged the fusion behavior of human neurospheres to improve upon this technique with our Neurosphere-derived organoid-like aggregates (NEDAS) culture method, allowing the fusion of human NSCs at high density, which were maintained in orbital shaker conditions for 8-12 weeks without passing leading to the formation of 3D organoid-like aggregates without the use of Matrigel. NEDAS organoids proliferate and self-organize into neural rosettes, expressing PAX6 and SOX2 in ventricular zone (VZ)-like proliferative areas. Outside these rosettes, we identified corridors of migratory radial glial progenitors expressing Phospo-vimentin, CRYAB. In addition to DLX2, CXCR4 + progenitors. Further, we found immature neurons within cortical-like areas highly enriched for DCX and TUJ1, in addition to GABA+ and excitatory VGLUT1+ neurons. Here, we provide a protocol to generate NEDAS, additionally, we present a protocol for immunostaining of NEDAS organoids for confocal imaging. This protocol may be useful to dissect the self-organization and morphogenetic programs of populations of human NSCs offering an advantageous alternative to the conventional neurospheres method, generating more cell type diversity, within tissue-like aggregates over extended periods of time without dissociation or passing. NEDAS may be a complementary method to cerebral organoids protocols from IPSCs. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation and expansion of cultures of human neural stem cells in reduced growth factor basement matrix Basic Protocol 2: Formation and fusion of neurospheres derived matrigel-free organoid-like aggregates (NEDAS) Basic Protocol 3: Harvest, cryosection, and imaging protocol for NEDAS.

Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation.

In demyelinating diseases including multiple sclerosis (MS), neural stem cells (NSCs) can replace damaged oligodendrocytes if the local microenvironment supports the required differentiation process. Although chitinase-like proteins (CLPs) form part of this microenvironment, their function in this differentiation process is unknown. Here, we demonstrate that murine Chitinase 3-like-3 (Chi3l3/Ym1), human Chi3L1 and Chit1 induce oligodendrogenesis. In mice, Chi3l3 is highly expressed in the subventricular zone, a stem cell niche of the adult brain, and in inflammatory brain lesions during experimental autoimmune encephalomyelitis (EAE). We find that silencing Chi3l3 increases severity of EAE. We present evidence that in NSCs Chi3l3 activates the epidermal growth factor receptor (EGFR), thereby inducing Pyk2-and Erk1/2- dependent expression of a pro-oligodendrogenic transcription factor signature. Our results implicate CLP-EGFR-Pyk2-MEK-ERK as a key intrinsic pathway controlling oligodendrogenesis.

Elevated expression of granulocyte-macrophage colony-stimulating factor receptor in multiple sclerosis lesions.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease that disproportionately affects young adults, leading to disability and high costs to society. Infiltration of T cells and monocytes into the central nervous system (CNS) is critical for disease initiation and progression. However, despite a great deal of effort the molecular mechanisms by which immune cells initiate and perpetuate CNS damage in MS have not yet been elucidated. In experimental autoimmune encephalomyelitis (EAE), an animal model of MS, granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by pathogenic Th1 and Th17 cells is critical for the recruitment of monocytes into the CNS during the initial stage of disease. We and others have recently shown that, compared with healthy individuals, MS patients have greater numbers of CD4+ and CD8+ T cells that produce GM-CSF. Here, we describe the expression of GM-CSF and its receptor, GM-CSFR, in normal brain and MS lesions. Our data show that in acute and chronic MS lesions, microglia and astrocytes have upregulated expression of GM-CSFR; in addition, we show that GM-CSF-associated molecules are also upregulated in MS lesions. These findings further strengthen the argument that GM-CSF signaling contributes to MS pathogenesis.

iPhemap: an atlas of phenotype to genotype relationships of human iPSC models of neurological diseases.

Disease modeling with induced pluripotent stem cells (iPSCs) is creating an abundance of phenotypic information that has become difficult to follow and interpret. Here, we report a systematic analysis of research practices and reporting bias in neurological disease models from 93 published articles. We find heterogeneity in current research practices and a reporting bias toward certain diseases. Moreover, we identified 663 CNS cell-derived phenotypes from 243 patients and 214 controls, which varied by mutation type and developmental stage in vitro We clustered these phenotypes into a taxonomy and characterized these phenotype-genotype relationships to generate a phenogenetic map that revealed novel correlations among previously unrelated genes. We also find that alterations in patient-derived molecular profiles associated with cellular phenotypes, and dysregulated genes show predominant expression in brain regions with pathology. Last, we developed the iPS cell phenogenetic map project atlas (iPhemap), an open submission, online database to continually catalog disease phenotypes. Overall, our findings offer new insights into the phenogenetics of iPSC-derived models while our web tool provides a platform for researchers to query and deposit phenotypic information of neurological diseases.

Clinical significance of an easy-to-use dual task for assessing inattention.

Purpose To investigate clinical significance of a newly developed paper-and-pencil type dual-task (Oiso-DT) for assessing inattention of brain-damaged patients. Methods A total of 134 healthy individuals and 44 patients with traumatic brain injury (TBI) were the subjects. Oiso-DT combined a cancelation sub-task and a written calculation sub-task on paper. The performance was scored based on the correct rate (CR) and success rate in the cancelation sub-task, and the number of correct answers and CR in the calculation sub-task for three minutes. Performances of Clinical Assessment for Attention (CAT) developed by The Japan Society for Higher Brain Dysfunction were also measured in TBI patients. Results Based on a simple and unique definition of cutoff values, abnormal performance was detected more often in the Oiso-DT than in any sub-task of CAT including Symbol Digit Modalities Test, The Memory Updating Test and Paced Auditory Serial Addition Task, etc. although the specificity was comparable. Conclusion This easy-to-use Oiso-DT might be valuable and sensitive for detecting inattention including mild deficit. Implications for Rehabilitation The Oiso-DT, a paper-and-pencil clinical test for assessing inattention demonstrates higher sensitivity for traumatic-brain-injury patients than standard neuropsychological tests. The task performance is evaluated by a simple and unique method without specific equipment or even a personal computer. The Oiso-DT might be valuable for detecting inattention including mild deficit. The Oiso-DT is easily applicable to patients with severe inattention who failed to complete complex tasks like paced auditory serial addition task.

Concise review: modeling multiple sclerosis with stem cell biological platforms: toward functional validation of cellular and molecular phenotypes in inflammation-induced neurodegeneration.

In recent years, tremendous progress has been made in identifying novel mechanisms and new medications that regulate immune cell function in multiple sclerosis (MS). However, a significant unmet need is the identification of the mechanisms underlying neurodegeneration, because patients continue to manifest brain atrophy and disability despite current therapies. Neural and mesenchymal stem cells have received considerable attention as therapeutic candidates to ameliorate the disease in preclinical and phase I clinical trials. More recently, progress in somatic cell reprogramming and induced pluripotent stem cell technology has allowed the generation of human "diseased" neurons in a patient-specific setting and has provided a unique biological tool that can be used to understand the cellular and molecular mechanisms of neurodegeneration. In the present review, we discuss the application and challenges of these technologies, including the generation of neurons, oligodendrocytes, and oligodendrocyte progenitor cells (OPCs) from patients and novel stem cell and OPC cellular arrays, in the discovery of new mechanistic insights and the future development of MS reparative therapies.

Pathogenesis of autosomal dominant hereditary spastic paraplegia (SPG6) revealed by a rat model.

Hereditary spastic paraplegias (HSPs) are characterized by progressive spasticity and weakness in the lower extremities that result from length-dependent central to peripheral axonal degeneration. Mutations in the non-imprinted Prader-Willi/Angelman syndrome locus 1 (NIPA1) transmembrane protein cause an autosomal dominant form of HSP (SPG6). Here, we report that transgenic (Tg) rats expressing a human NIPA1/SPG6 mutation in neurons (Thy1.2-hNIPA1) show marked early onset behavioral and electrophysiologic abnormalities. Detailed morphologic analyses reveal unique histopathologic findings, including the accumulation of tubulovesicular organelles with endosomal features that start at axonal and dendritic terminals, followed by multifocal vacuolar degeneration in both the CNS and peripheral nerves. In addition, the NIPA1 mutation in the spinal cord from older Tg rats results in an increase in bone morphogenetic protein type II receptor expression, suggesting that its degradation is impaired. This Thy1.2-hNIPA1 Tg rat model may serve as a valuable tool for understanding endosomal trafficking in the pathogenesis of a subgroup of HSP with an abnormal interaction with bone morphogenetic protein type II receptor, as well as for developing potential therapeutic strategies for diseases with axonal degeneration and similar pathogenetic mechanisms.

Surgical procedure and results of cisternal washing therapy for the prevention of cerebral vasospasm following SAH.

In 1994, we started cisternal washing therapy (CWT) using urokinase combined with head-shaking method in order to prevent cerebral vasospasm. In this paper, we showed the surgical procedure for CWT and reported the effect of this therapy in preventing vasospasm following SAH. A total of 332 consecutive cases with Fisher group 3 SAH since 1988 were analyzed. Of these patients, 118 cases (56 cases before 1994 and 62 cases after 1994) had not CWT, and, 214 cases after 1994 had this therapy. All of these patients had clipping surgery within 3 days following SAH, and had postoperative management both with normovolemia and normal to mild hypertension. In these two groups, the incidence of symptomatic vasospasm (transiently symptomatic vasospasm without infarction), cerebral infarction due to vasospasm on CT, and mortality and morbidity (M&M) due to vasospasm were analyzed. In the group without CWT, the incidences of symptomatic vasospasm, cerebral infarction on CT, and M&M due to vasospasm were 4.2%, 28.8%, and 17.8%, respectively. On the other hand, in the group with CWT, they were 3.7%, 6.5%, and 2.8%, respectively. In the patients with CWT, the incidence of cerebral infarction on CT due to vasospasm and M&M due to vasospasm were significantly (p < 0.05) decreased. CWT was effective in preventing cerebral vasospasm.

Effects of FAK ablation on cerebellar foliation, Bergmann glia positioning and climbing fiber territory on Purkinje cells.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is widely expressed in the brain, and plays key roles in various cellular processes in response to both extracellular and intracellular stimuli. Here, we explored the role of FAK in cerebellar development. In the mouse cerebellum, FAK was found to be distributed as tiny cytoplasmic aggregates in various neuronal and glial elements, including Purkinje cells (PCs), Bergmann glia (BG), parallel fiber (PF)-terminals and climbing fiber (CF)-terminals. The neuron/glia-specific ablation of FAK impaired cerebellar foliation, such as variable decreases in foliation sizes and the lack of intercrural and precentral fissures. Some of the BG cells became situated ectopically in the molecular layer. Furthermore, the FAK ablation altered the innervation territories of CFs and PFs on PCs. CF innervation regressed to the basal portion of proximal dendrites and somata, whereas ectopic spines protruded from proximal dendrites and PFs expanded their territory by innervating the ectopic spines. Furthermore, the persistence of surplus CFs innervating PC somata caused multiple innervation. When FAK was selectively ablated in PCs, diminished dendritic innervation and persistent somatic innervation by CFs were observed, whereas cerebellar foliation and cell positioning of BG were normally retained. These results suggest that FAK in various neuronal and glial elements is required for the formation of normal histoarchitecture and cytoarchitecture in the cerebellum, and for the construction of proper innervation territory and synaptic wiring in PCs.

Intermanual difference in the Japanese Trail Making Test and its mirror version: intra-subject comparison of the task-completion time, cognitive time, and motor time.

PURPOSE: To investigate intermanual differences in the performance of the Japanese Trail Making Test (JTMT) using an intra-subject comparison, and to investigate how the cognitive and motor times spent in performing the task were related with the total completion time. METHODS: All subjects performed both parts A and B of the following three tasks: JTMT with the right hand (R); JTMT with the left hand (L); and a mirror version of the JTMT with the left hand (M). The order in which these three tasks were performed (RLM, RML, LRM, LMR, MRL, and MLR) was randomly determined. An interval of 4 weeks was scheduled between each examination. In addition to the completion time, we measured 'motor time', a sum of the times for drawing lines to connect the targets, and 'cognitive time', a time obtained by subtracting the motor time from the completion time. RESULTS: The task-completion time was more strongly correlated with the cognitive time than with the motor time. The order of the task performance did not influence the task-completion time. A learning effect was found only between the first and second examinations of part A. The cognitive time for the second and third performances of part A were significantly shorter than that for the first task, whereas the motor time remained unchanged. No intermanual differences in the performance were found in any comparisons. CONCLUSIONS: The non-dominant hand can be used as an alternative hand in the JTMT, and it can be expected to perform comparably to the dominant hand. These findings are consistent with the finding previously obtained by inter-subject comparison. The lack of any significant intermanual difference can be explained by that the total completion time is less influenced by the motor time than by the cognitive time.