Early Neural Stem Cells Research Articles

MDB-09. IMPACT OF PRDM6 ON CHROMATIN ACCESSIBILITY, GENE EXPRESSION, AND MEDULLOBLASTOMA FORMATION

Abstract Group 4 medulloblastoma (MB) is the most common medulloblastoma subgroup and shows high incidence of metastasis and late-onset relapse cases. These tumors lack a unifying oncogenic driver and treatment targets, despite extensive genomic characterization. Group 4 MBs are characterized by recurrent genetic alterations in chromatin modifiers and amplification of stemness genes. A substantial fraction of Group 4 MBs are characterized by enhancer hijacking through tandem duplication of SNCAIP, resulting in high expression of PRDM6, a putative transcriptional repressor and histone methyltransferase. Some PRDM6-overexpressing medulloblastoma cases show additional mutations in chromatin regulators and high MYCN expression. We set out to elucidate the impact and oncogenic potential of sustained PRDM6 expression in early neural stem cell populations and patient-derived medulloblastoma cells. We find that PRDM6 expression in human iPSC-derived neuroepithelial stem cells (NESCs) results in tumor growth in mice at low penetrance. Moreover, we find that PRDM6 expression in MYCN-overexpressing NESCs does not further alter tumor aggressiveness or survival in vivo. Notably, PRDM6 overexpression in the patient-derived Group 3 MYC-amplified D283-Med cell line causes significantly increased aggressiveness of tumor growth and shorter survival in mice. At the cellular level, PRDM6 localizes to the nucleus, suggesting a role in gene expression regulation. Consistent with this notion, ATAC-seq and RNA-seq analysis of PRDM6-NESCs and PRDM6-D283-Med cells reveals major changes in chromatin accessibility and gene expression, specifically upregulation of integrin family members and gene family members of focal adhesion and extracellular matrix receptor interaction pathways. We conclude that PRDM6 promotes tumor growth in NESCs and may play a role in tumor maintenance through interactions with the extracellular matrix and tumor microenvironment.

Pipsqueak family genes dan/danr antagonize nuclear Pros to prevent neural stem cell aging in Drosophila larval brains.

Neural stem cell aging is a fundamental question in neurogenesis. Premature nuclear Pros is considered as an indicator of early neural stem cell aging in Drosophila. The underlying mechanism of how neural stem cells prevent premature nuclear Pros remains largely unknown. Here we identified that two pipsqueak family genes, distal antenna (dan) and distal antenna-related (danr), promote the proliferation of neural stem cells (also called neuroblasts, NBs) in third instar larval brains. In the absence of Dan and Danr (dan/danr), the NBs produce fewer daughter cells with smaller lineage sizes. The larval brain NBs in dan/danr clones show premature accumulation of nuclear Prospero (Pros), which usually appears in the terminating NBs at early pupal stage. The premature nuclear Pros leads to NBs cell cycle defects and NB identities loss. Removal of Pros from dan/danr MARCM clones prevents lineage size shrinkage and rescues the loss of NB markers. We propose that the timing of nuclear Pros is after the downregulation of dan/danr in the wt terminating NBs. dan/danr and nuclear Pros are mutually exclusive in NBs. In addition, dan/danr are also required for the late temporal regulator, Grainyhead (Grh), in third instar larval brains. Our study uncovers the novel function of dan/danr in NBs cell fate maintenance. dan/danr antagonize nuclear Pros to prevent NBs aging in Drosophila larval brains.

MODL-32. IMPACT OF PRDM6 ON CHROMATIN ACCESSIBILITY, GENE EXPRESSION, AND MEDULLOBLASTOMA FORMATION

Abstract Group 4 medulloblastoma (MB) is the most common medulloblastoma subgroup and shows high incidence of metastasis and late-onset relapse. Group 4 MBs lack a unifying oncogenic driver and treatment targets, despite extensive genomic characterization. Group 4 MBs are characterized by recurrent genetic alterations in chromatin modifiers, amplification of stemness genes, and putative enhancer "hijacking" events. A substantial fraction of Group 4 MBs are characterized by enhancer hijacking through tandem duplication of SNCAIP, resulting in high expression of PRDM6, a putative transcriptional repressor and histone methyltransferase. Some PRDM6-overexpressing MBs show additional mutations in chromatin regulators and high MYCN expression. We set out to elucidate the impact and oncogenic potential of sustained PRDM6 expression in early neural stem cell populations and patient-derived medulloblastoma cells. We find that PRDM6 expression in human iPSC-derived neuroepithelial stem cells (NESCs) results in tumor growth in mice, albeit at low penetrance. Moreover, we find that PRDM6 expression in MYCN-overexpressing NESCs does not further alter tumor aggressiveness or survival in vivo. Notably, PRDM6 overexpression in the patient-derived Group 3 MYC-amplified D283-Med cell line causes significantly increased aggressiveness of tumor growth and shorter survival in mice. At the cellular level, PRDM6 localizes to the nucleus, suggesting a role in gene expression regulation. Consistent with this notion, ATAC-seq and RNA-seq analysis of PRDM6-NESCs and PRDM6-D283-Med cells reveals major changes in chromatin accessibility and gene expression, including upregulation of integrin family members and genes related to focal adhesion and extracellular matrix-receptor interaction pathways. We conclude that PRDM6 promotes tumor growth in NESCs and may play a role in tumor maintenance through interactions with the extracellular matrix and tumor microenvironment.

Enhanced cortical neural stem cell identity through short SMAD and WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells

Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity despite the tremendous upsurge in derivation methods, suggesting inadequate patterning of early neural stem cells (NSCs). Here we show that a short and early Dual SMAD and WNT inhibition course is necessary and sufficient to establish robust and lasting cortical organoid NSC identity, efficiently suppressing non-cortical NSC fates, while other widely used methods are inconsistent in their cortical NSC-specification capacity. Accordingly, this method selectively enriches for outer radial glia NSCs, which cyto-architecturally demarcate well-defined outer sub-ventricular-like regions propagating from superiorly radially organized, apical cortical rosette NSCs. Finally, this method culminates in the emergence of molecularly distinct deep and upper cortical layer neurons, and reliably uncovers cortex-specific microcephaly defects. Thus, a short SMAD and WNT inhibition is critical for establishing a rich cortical cell repertoire that enables mirroring of fundamental molecular and cyto-architectural features of cortical development and meaningful disease modelling.

A comparative study of the capability of MSCs isolated from different human tissue sources to differentiate into neuronal stem cells and dopaminergic-like cells.

BackgroundNeurodegenerative diseases are characterized by progressive neuronal loss and degeneration. The regeneration of neurons is minimal and neurogenesis is limited only to specific parts of the brain. Several clinical trials have been conducted using Mesenchymal Stem Cells (MSCs) from different sources to establish their safety and efficacy for the treatment of several neurological disorders such as Parkinson’s disease, multiple sclerosis and amyotrophic lateral sclerosis.AimThe aim of this study was to provide a comparative view of the capabilities of MSCs, isolated from different human tissue sources to differentiate into neuronal stem cell-like cells (NSCs) and possibly into dopaminergic neural- like cells.MethodsMesenchymal stem cells were isolated from human bone marrow, adipose, and Wharton’s jelly (WJ) tissue samples. Cells were characterized by flow cytometry for their ability to express the most common MSC markers. The differentiation potential was also assessed by differentiating them into osteogenic and adipogenic cell lineages. To evaluate the capacity of these cells to differentiate towards the neural stem cell-like lineage, cells were cultured in media containing small molecules. Cells were utilized for gene expression and immunofluorescence analysis at different time points.ResultsOur results indicate that we have successfully isolated MSCs from bone marrow, adipose tissue, and Wharton’s jelly. WJ-MSCs showed a slightly higher proliferation rate after 72 hours compared to BM and AT derived MSCs. Gene expression of early neural stem cell markers revealed that WJ-MSCs had higher expression of Nestin and PAX6 compared to BM and AT-MSCs, in addition to LMX expression as an early dopaminergic neural marker. Immunofluorescence analysis also revealed that these cells successfully expressed SOX1, SOX2, Nestin, TUJ1, FOXA2 and TH.ConclusionThese results indicate that the protocol utilized has successfully differentiated BM, AT and WJ-MSCs into NSC-like cells. WJ-MSCs possess a higher potential to transdifferentiate into NSC and dopaminergic-like cells. Thus, it might indicate that this protocol can be used to induce MSC into neuronal lineage, which provides an additional or alternative source of cells to be used in the neurological cell-based therapies.

BIOL-11. THE ROLE OF ABERRANT EXPRESSION OF PRDM6 IN THE DEVELOPING CEREBELLUM AND IN GROUP 4 MEDULLOBLASTOMA

Group 4 medulloblastoma is the most common medulloblastoma subgroup with an intermediate prognosis and a high incidence of metastasis and late-onset relapse cases. Despite several comprehensive genomic studies in medulloblastoma, Group 4 medulloblastomas lack a unifying oncogenic driver and treatment targets. This subgroup is characterized by recurrent genetic alterations in chromatin modifiers, amplification of stemness genes, and enhancer hijacking events. 17% of Group 4 medulloblastoma cases are characterized by enhancer hijacking through tandem duplication of SNCAIP, resulting in high expression of PRDM6, a putative transcriptional repressor and histone methyltransferase. PRDM6 amplified medulloblastoma cases show additional mutations in other chromatin regulators, such as KDM6A, KMT2C and KMT2D, ZMYM3, and high MYCN expression. In this project, we investigate the impact and oncogenic potential of sustained PRDM6 expression in early neural stem cell populations and the developing mouse cerebellum. We drive expression of PRDM6 in human iPSC-derived neuroepithelial stem cells (NESCs) with and without high MYCN expression to study its implications in tumorigenesis. To test for tumor growth in vivo and changes in tumor progression as a function of PRDM6 activity, NESCs are injected into the cerebellum of adult mice. In order to elucidate impact of PRDM6 activity during embryonic cerebellar development, we also introduce PRDM6 expression into mouse embryonic stem cells (ESCs) for analysis via a new, in vivo cerebellar blastocyst complementation model. The latter approach is designed to ablate and repopulate early granule neural precursor cells in the embryonal cerebellum with progenitors derived from injected PRDM6-ESCs and thus to recapitulate pre- and postnatal cerebellar development in vivo. Together, our studies aim to understand the role of PRDM6 during normal cerebellar development and tumorigenesis and advance the understanding of the genetic drivers for Group 4 medulloblastoma.

Dissecting the impact of regional identity and the oncogenic role of human-specific NOTCH2NL in an hESC model of H3.3G34R-mutant glioma

H3.3G34R-mutant gliomas are lethal tumors of the cerebral hemispheres with unknown mechanisms of regional specificity and tumorigenicity. We developed a human embryonic stem cell (hESC)-based model of H3.3G34R-mutant glioma that recapitulates the key features of the tumors with cell-type specificity to forebrain interneuronal progenitors but not hindbrain precursors. We show that H3.3G34R, ATRX, and TP53 mutations cooperatively impact alternative RNA splicing events, particularly suppression of intron retention. This leads to increased expression of components of the Notch pathway, notably NOTCH2NL, a human-specific gene family. We also uncover a parallel mechanism of enhanced NOTCH2NL expression via genomic amplification of its locus in some H3.3G34R-mutant tumors. These findings demonstrate a novel mechanism whereby evolutionary pathways that lead to larger brain size in humans are co-opted to drive tumor growth.

Generation of brain organoids from mouse ESCs via teratoma formation.

Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs), can differentiate into all cell types in the body; therefore, they are used in the study of development and regenerative medicine. Neural lineage differentiation from PSCs is the initial step to study neurodevelopment and in vitro disease modeling. Brain organoids, which are composed of neural stem cells (NSCs) and differentiated neural lineage cell population, are a powerful in vitro system to mimic the brain tissue. Here, we aimed to establish a new method to generate brain organoids efficiently in a mouse model. We applied the in vivo teratoma formation method as a new approach to generate brain organoids. We induced teratoma formation using Sox1-GFP transgenic ESCs, in which green fluorescence protein (GFP) is expressed under the control of the early NSC marker Sox1. Sox1-GFP-expressing early NSCs were isolated as clumps and further cultured to generate brain organoids. Sox1-GFP ESC-derived brain organoids, composed of multiple layers of distinct cellular components (ventricle, ventricular zone, and cortical layer), were formed within 3weeks of in vitro culture. We also found that neighboring cells (Sox1-GFP-) surrounding the Sox1-GFP+ clumps are essential for the formation of brain organoids. Thus, in vivo and in vitro conjugated systems-initial commitment in vivo and further specialization in vitro-could be one of the promising platforms for organoid formation that are universally applicable.

Pre-clinical study of induced pluripotent stem cell-derived dopaminergic progenitor cells for Parkinson\u2019s disease

Induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neurons are an expected source for cell-based therapies for Parkinson’s disease (PD). The regulatory criteria for the clinical application of these therapies, however, have not been established. Here we show the results of our pre-clinical study, in which we evaluate the safety and efficacy of dopaminergic progenitors (DAPs) derived from a clinical-grade human iPSC line. We confirm the characteristics of DAPs by in vitro analyses. We also verify that the DAP population include no residual undifferentiated iPSCs or early neural stem cells and have no genetic aberration in cancer-related genes. Furthermore, in vivo studies using immunodeficient mice reveal no tumorigenicity or toxicity of the cells. When the DAPs are transplanted into the striatum of 6-OHDA-lesioned rats, the animals show behavioral improvement. Based on these results, we started a clinical trial to treat PD patients in 2018.

Hypoxia/Hif1α prevents premature neuronal differentiation of neural stem cells through the activation of Hes1.

Embryonic neural stem cells (NSCs), comprising neuroepithelial and radial glial cells, are indispensable precursors of neurons and glia in the mammalian developing brain. Since the process of neurogenesis occurs in a hypoxic environment, the question arises of how NSCs deal with low oxygen tension and whether it affects their stemness. Genes from the hypoxia-inducible factors (HIF) family are well known factors governing cellular response to hypoxic conditions. In this study, we have discovered that the endogenous stabilization of hypoxia-inducible factor 1α (Hif1α) during neural induction is critical for the normal development of the NSCs pool by preventing its premature depletion and differentiation. The knock-out of the Hif1α gene in mESC-derived neurospheres led to a decrease in self-renewal of NSCs, paralleled by an increase in neuronal differentiation. Similarly, neuroepithelial cells differentiated in hypoxia exhibited accelerated neurogenesis soon after Hif1α knock-down. In both models, the loss of Hif1α was accompanied by an immediate drop in neural repressor Hes1 levels while changes in Notch signaling were not observed. We found that active Hif1α/Arnt1 transcription complex bound to the evolutionarily conserved site in Hes1 gene promoter in both neuroepithelial cells and neural tissue of E8.5 - 9.5 embryos. Taken together, these results emphasize the novel role of Hif1α in the regulation of early NSCs population through the activation of neural repressor Hes1, independently of Notch signaling.

BMP Inhibition in the Presence of LIF Differentiates Murine Embryonic Stem Cells to Early Neural Stem Cells

Early mouse neural stem cells (NSCs) first appear in embryonic day E5.5 and express pluripotency markers Oct4, Sox2, Nanog and early neural marker Sox1. Early NSCs are a good model for understanding the role of various pathways that control initial neural commitment. However, a protocol for differentiation of mouse embryonic stem cells (ESCs) into early NSCs by adherent monolayer culture has not yet been established. Hence, in this study, we identified the combination of growth factors and small molecules that differentiated mouse ESCs into early NSCs and supported their proliferation. Leukaemia inhibitory factor (LIF) was the first factor to be tested and it was found that ESCs can differentiate into early neurogenic lineage in the presence of LIF. However, we found that the induction is weaker in the presence of LIF as compared to cells differentiated in its absence. GSK-3 inhibitor, along with BMP and TGF-β pathway inhibitor (dual SMAD inhibition), are commonly used to sequentially direct ESCs towards NSCs. However, when we used this combination, mouse ESCs failed to differentiate into early NSCs. We observed that by adding Wnt inhibitor to the combination of GSK-3 inhibitor, BMP inhibitor, TGF-β inhibitor and LIF, it was possible to differentiate ESCs into early NSCs. qRT-PCR analysis of early NSCs illustrated that they expressed key pluripotency genes Oct4 and Nanog, albeit at levels lower than non-differentiated ESCs, along with early neural markers Sox1 and Pax6.

A Nucleolar Protein, Nepro, Is Essential for the Maintenance of Early Neural Stem Cells and Preimplantation Embryos.

Notch signaling is required for maintaining neural stem cells (NSCs) in the developing brain. NSCs have potential to give rise to many neuronal types in the early telencephalon, and the potential decreases as embryonic development proceeds. Nepro, which encodes a unique nucleolar protein and is activated downstream of Notch, is essential for maintaining NSCs in the early telencephalon. Nepro is also expressed at basal levels and required for maintaining the preimplantation embryo, by repressing mitochondria-associated p53 apoptotic signaling. Notch signaling also controls dendritic complexity in mitral cells, major projection neurons in the olfactory bulb, showing that many steps of neural development involve Notch signaling.

SOX19b regulates the premature neuronal differentiation of neural stem cells through EZH2-mediated histone methylation in neural tube development of zebrafish

ObjectiveNeural tube defects (NTDs) are the most serious and common birth defects in the clinic. The SRY-related HMG box B1 (SoxB1) gene family has been implicated in different processes of early embryogenesis. Sox19b is a maternally expressed gene in the SoxB1 family that is found in the region of the presumptive central nervous system (CNS), but its role and mechanism in embryonic neural stem cells (NSCs) during neural tube development have not yet been explored. Considering that Sox19b is specific to bony fish, we intended to investigate the role and mechanism of Sox19b in neural tube development in zebrafish embryos.Material and methodsMorpholino (MO) antisense oligonucleotides were used to construct a Sox19b loss-of-function zebrafish model. The phenotype and the expression of related genes were analysed by in situ hybridization and immunolabelling. Epigenetic modifications were detected by western blot and chromatin immunoprecipitation.ResultsIn this study, we found that zebrafish embryos exhibited a reduced or even deleted forebrain phenotype after the expression of the Sox19b gene was inhibited. Moreover, we found for the first time that knockdown of Sox19b reduced the proliferation of NSCs; increased the transcription levels of Ngn1, Ascl1, HuC, Islet1, and cyclin-dependent kinase (CDK) inhibitors; and led to premature differentiation of NSCs. Finally, we found that knockdown of Sox19b decreased the levels of EZH2/H3K27me3 and decreased the level of H3K27me3 at the promoters of Ngn1 and ascl1a.ConclusionTogether, our data demonstrate that Sox19b plays an essential role in early NSC proliferation and differentiation through EZH2-mediated histone methylation in neural tube development. This study established the role of transcription factor Sox19b and epigenetic factor EZH2 regulatory network on NSC development, which provides new clues and theoretical guidance for the clinical treatment of neural tube defects.

PDTM-22. A C19MC-LIN28A-MYCN ONCOGENIC CIRCUIT DRIVEN BY HIJACKED SUPER-ENHANCERS IS A DISTINCT THERAPEUTIC VULNERABILITY IN ETMRS – A LETHAL BRAIN TUMOR

Abstract Embryonal tumors with multi-layered rosettes (ETMR) are aggressive brain cancers with characteristic C19MC oncomiR amplification and enrichment of pluripotency factor LIN28A. Here we investigated C19MC oncogenic mechanisms and discovered a potent C19MC-LIN28A-MYCN circuitry driven by multiple regulatory loops and super-enhancers resulting from long-range MYCN DNA interactions and C19MC gene fusions. C19MC and LIN28A targets respectively converge on critical cell cycle tumor suppressors and neo-embryonic DNMT3A/B isoforms. We identify a MYCN driven, core transcriptional network, conserved in early neural stem cells, that is potently abrogated by treatment with bromodomain inhibitor JQ1, leading to ETMR cell death. Our collective data suggest C19MC oncomiRs drive a malignant primitive cell state in ETMRs via entrapment of an early neural lineage network, which represents a critical therapeutic vulnerability for this recalcitrant disease.

Toxicity of ionizing radiation (IR) in a human induced pluripotent stem cell (hiPSC)-derived 3D early neurodevelopmental model.

Prenatal brain development is a complex and sensitive process, highly susceptible to environmental influences such as pollutants, stress, malnutrition, drugs, tobacco exposure, or ionizing radiation (IR). Disturbances in development may cause life-long disabilities and diseases, such as ADHD, childhood cancers, cognitive problems, depression, anxiety and more severe developmental disabilities. Due to increasing medical imaging, radiation therapy, natural terrestrial radiation, radioactive pollution and long-distance flights, humans are increasingly exposed to IR. However, data on impact of IR on very early human brain development are scarce, particularly in the very first weeks of gestation. Here we investigated the effects of low-dose X-ray IR (1Gy) in a 3D early brain developmental model derived from human pluripotent stem cells. In this model very early neural stem cells, neuroectodermal progenitor cells (NEP), were exposed to low-dose IR and direct as well as delayed effects were investigated. Expression of 20 different marker genes crucial for normal neural development was determined 48h and 9days post IR (pIR). All but one of the analyzed marker genes were reduced 48h after IR, and all but seven genes normalized their expression by day 9 pIR. Among the seven markers were genes involved in neurodevelopmental and growth abnormalities. Moreover, we could show that stemness of the NEP was reduced after IR. We were thus able to identify a significant impact of radiation in cells surviving low-dose IR, suggesting that low-dose IR could have a negative impact on the early developing human brain, with potential later detrimental effects.

Surfaceome Profiling Reveals Regulators of Neural Stem Cell Function

The composition of cell-surface proteins changes during lineage specification, altering cellular responses to their milieu. The changes that characterize maturation of early neural stem cells (NSCs) remain poorly understood. Here we use mass spectrometry-based cell surface capture technology to profile the cell surface of early NSCs and demonstrate functional requirements for several enriched molecules. Primitive NSCs arise from embryonic stem cells upon removal of Transforming growth factor-β signaling, while definitive NSCs arise from primitive NSCs upon Lif removal and FGF addition. In vivo aggregation assays revealed that N-cadherin upregulation is sufficient for the initial exclusion of definitive NSCs from pluripotent ectoderm, while c-kit signaling limits progeny of primitive NSCs. Furthermore, we implicate EphA4 in primitive NSC survival signaling and Erbb2 as being required for NSC proliferation. This work elucidates several key mediators of NSC function whose relevance is confirmed on forebrain-derived populations and identifies a host of other candidates that may regulate NSCs.

Shifts in the vascular endothelial growth factor isoforms result in transcriptome changes correlated with early neural stem cell proliferation and differentiation in mouse forebrain

Regulation of neural stem cell (NSC) fate decisions is critical during the transition from a multicellular mammalian forebrain neuroepithelium to the multilayered neocortex. Forebrain development requires coordinated vascular investment alongside NSC differentiation. Vascular endothelial growth factor A (Vegf) has proven to be a pleiotrophic gene whose multiple protein isoforms regulate a broad range of effects in neurovascular systems. To test the hypothesis that the Vegf isoforms (120, 164, and 188) are required for normal forebrain development, we analyzed the forebrain transcriptome of mice expressing specific Vegf isoforms, Vegf120, VegfF188, or a combination of Vegf120/188. Transcriptome analysis identified differentially expressed genes in embryonic day (E) 9.5 forebrain, a time point preceding dramatic neuroepithelial expansion and vascular investment in the telencephalon. Meta-analysis identified gene pathways linked to chromosome-level modifications, cell fate regulation, and neurogenesis that were altered in Vegf isoform mice. Based on these gene network shifts, we predicted that NSC populations would be affected in later stages of forebrain development. In the E11.5 telencephalon, we quantified mitotic cells [Phospho-Histone H3 (pHH3)-positive] and intermediate progenitor cells (Tbr2/Eomes-positive), observing quantitative and qualitative shifts in these populations. We observed qualitative shifts in cortical layering at P0, particularly with Ctip2-positive cells in layer V. The results identify a suite of genes and functional gene networks that can be used to further dissect the role of Vegf in regulating NSC differentiation and downstream consequences for NSC fate decisions.

Recruitment of neural precursor cells from circumventricular organs of patients with cerebral ischaemia

Adult neurogenesis is well described in the subventricular zone of the lateral ventricle walls and in the subgranular zone of the hippocampal dentate gyrus. However, recent studies indicate that self-renewal of neural stem cells (NSCs) is not restricted to these niches, but that diverse areas of the adult brain are capable of generating new neurones and responding to various pathological alterations. In particular, NSCs have been identified in circumventricular organs (CVOs) of the adult mouse brain. In order to detect possible neural stem or progenitor cells in CVOs of the human brain, we analysed post mortem human brain tissue from patients without neuropathological changes (n = 16) and brains from patients with ischaemic stroke (n = 16). In all analysed CVOs (area postrema, median eminence, pineal gland and neurohypophysis) we observed cells with expression of early NSC markers, such as GFAP, nestin, vimentin, OLIG2 and PSA-NCAM, with some of them coexpressing Ki67 as a marker of cell proliferation. Importantly, stroke patients displayed an up to fivefold increase with respect to the relative number of Ki67- and OLIG2-expressing cells within their CVOs. Our findings are compatible with a scenario where CVOs may serve as a further source of NSCs in the adult human brain and may contribute to neurogenesis and brain plasticity in the context of brain injury.

EZ spheres: A stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell–cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology.

Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells

Under appropriate culture conditions, bone marrow (BM)-derived mesenchymal stem cells are capable of differentiating into diverse cell types unrelated to their phenotypical embryonic origin, including neural cells. Here, we report the successful generation of neural stem cell (NSC)-like cells from BM-derived human mesenchymal stem cells (hMSCs). Initially, hMSCs were cultivated in a conditioned medium of human neural stem cells. In this culture system, hMSCs were induced to become NSC-like cells, which proliferate in neurosphere-like structures and express early NSC markers. Like central nervous system-derived NSCs, these BM-derived NSC-like cells were able to differentiate into cells expressing neural markers for neurons, astrocytes, and oligodendrocytes. Whole-cell patch clamp recording revealed that neuron-like cells, differentiated from NSC-like cells, exhibited electrophysiological properties of neurons, including action potentials. Transplantation of NSC-like cells into mouse brain confirmed that these NSC-like cells retained their capability to differentiate into neuronal and glial cells in vivo. Our data show that multipotent NSC-like cells can be efficiently produced from BM-derived hMSCs in culture and that these cells may serve as a useful alternative to human neural stem cells for potential clinical applications such as autologous neuroreplacement therapies.