Showing papers on "Neurosphere published in 2021"

m6A-RNA Demethylase FTO Inhibitors Impair Self-Renewal in Glioblastoma Stem Cells.

Abstract: N6-methyladenosine (m6A) has emerged as the most abundant mRNA modification that regulates gene expression in many physiological processes. m6A modification in RNA controls cellular proliferation and pluripotency and has been implicated in the progression of multiple disease states, including cancer. RNA m6A methylation is controlled by a multiprotein "writer" complex including the enzymatic factor methyltransferase-like protein 3 (METTL3) that regulates methylation and two "eraser" proteins, RNA demethylase ALKBH5 (ALKBH5) and fat mass- and obesity-associated protein (FTO), that demethylate m6A in transcripts. FTO can also demethylate N6,2'-O-dimethyladenosine (m6Am), which is found adjacent to the m7G cap structure in mRNA. FTO has recently gained interest as a potential cancer target, and small molecule FTO inhibitors such as meclofenamic acid have been shown to prevent tumor progression in both acute myeloid leukemia and glioblastoma in vivo models. However, current FTO inhibitors are unsuitable for clinical applications due to either poor target selectivity or poor pharmacokinetics. In this work, we describe the structure-based design, synthesis, and biochemical evaluation of a new class of FTO inhibitors. Rational design of 20 small molecules with low micromolar IC50's and specificity toward FTO over ALKBH5 identified two competitive inhibitors FTO-02 and FTO-04. Importantly, FTO-04 prevented neurosphere formation in patient-derived glioblastoma stem cells (GSCs) without inhibiting the growth of healthy neural stem cell-derived neurospheres. Finally, FTO-04 increased m6A and m6Am levels in GSCs consistent with FTO inhibition. These results support FTO-04 as a potential new lead for treatment of glioblastoma.

The SRSF1/circATP5B/miR-185-5p/HOXB5 feedback loop regulates the proliferation of glioma stem cells via the IL6-mediated JAK2/STAT3 signaling pathway

Abstract: Glioma is the most common and malignant tumor of central nervous system. The tumor initiation, self-renewal, and multi-lineage differentiation abilities of glioma stem cells (GSCs) are responsible for glioma proliferation and recurrence. Although circular RNAs (circRNAs) play vital roles in the progression of glioma, the detailed mechanisms remain unknown. qRT-PCR, western blotting, immunohistochemistry, and bioinformatic analysis were performed to detect the expression of circATP5B, miR-185-5p, HOXB5, and SRSF1. Patient-derived GSCs were established, and MTS, EDU, neurosphere formation, and limiting dilution assays were used to detect the proliferation of GSCs. RNA-binding protein immunoprecipitation, RNA pull-down, luciferase reporter assays, and chromatin immunoprecipitation assays were used to detect these molecules’ regulation mechanisms. We found circATP5B expression was significantly upregulated in GSCs and promoted the proliferation of GSCs. Mechanistically, circATP5B acted as miR-185-5p sponge to upregulate HOXB5 expression. HOXB5 was overexpressed in glioma and transcriptionally regulated IL6 expression and promoted the proliferation of GSCs via JAK2/STAT3 signaling. Moreover, RNA binding protein SRSF1 could bind to and promote circATP5B expression and regulate the proliferation of GSCs, while HOXB5 also transcriptionally regulated SRSF1 expression. Our study identified the SRSF1/circATP5B/miR-185-5p/HOXB5 feedback loop in GSCs. This provides an effective biomarker for glioma diagnosis and prognostic evaluation.

Neuronal Differentiation from Induced Pluripotent Stem Cell-Derived Neurospheres by the Application of Oxidized Alginate-Gelatin-Laminin Hydrogels.

Abstract: Biodegradable hydrogels that promote stem cell differentiation into neurons in three dimensions (3D) are highly desired in biomedical research to study drug neurotoxicity or to yield cell-containing biomaterials for neuronal tissue repair. Here, we demonstrate that oxidized alginate-gelatin-laminin (ADA-GEL-LAM) hydrogels facilitate neuronal differentiation and growth of embedded human induced pluripotent stem cell (hiPSC) derived neurospheres. ADA-GEL and ADA-GEL-LAM hydrogels exhibiting a stiffness close to ~5 kPa at initial cell culture conditions of 37 °C were prepared. Laminin supplemented ADA-GEL promoted an increase in neuronal differentiation in comparison to pristine ADA-GEL, with enhanced neuron migration from the neurospheres to the bulk 3D hydrogel matrix. The presence of laminin in ADA-GEL led to a more than two-fold increase in the number of neurospheres with migrated neurons. Our findings suggest that laminin addition to oxidized alginate-gelatin hydrogel matrices plays a crucial role to tailor oxidized alginate-gelatin hydrogels suitable for 3D neuronal cell culture applications.

PARK7 maintains the stemness of glioblastoma stem cells by stabilizing epidermal growth factor receptor variant III.

Abstract: PARK7 is involved in many key cellular processes, including cell proliferation, transcriptional regulation, cellular differentiation, oxidative stress protection, and mitochondrial function maintenance. Deregulation of PARK7 has been implicated in the pathogenesis of various human diseases, including cancer. Here, we aimed to clarify the effect of PARK7 on stemness and radioresistance of glioblastoma stem cells (GSCs). Serum differentiation and magnetic cell sorting of GSCs revealed that PARK7 was preferentially expressed in GSCs rather than differentiated GSCs. Immunohistochemical staining showed enhanced expression of PARK7 in glioma tissues compared to that in normal brain tissues. shRNA-mediated knockdown of PARK7 inhibited the self-renewal activity of GSCs in vitro, as evidenced by the results of neurosphere formation, limiting dilution, and soft-agar clonogenic assays. In addition, PARK7 knockdown suppressed GSC invasion and enhanced GSC sensitivity to ionizing radiation (IR). PARK7 knockdown suppressed expression of GSC signatures including nestin, epidermal growth factor receptor variant III (EGFRvIII), SOX2, NOTCH1, and OCT4. Contrarily, overexpression of PARK7 in CD133- non-GSCs increased self-renewal activities, migration, and IR resistance, and rescued the reduction of GSC factors under shPARK7-transfected and serum-differentiation conditions. Intriguingly, PARK7 acted as a co-chaperone of HSP90 by binding to it, protecting EGFRvIII from proteasomal degradation. Knockdown of PARK7 increased the production of reactive oxygen species, inducing partial apoptosis and enhancing IR sensitivity in GSCs. Finally, PARK7 knockdown increased mouse survival and IR sensitivity in vivo. Based on these data, we propose that PARK7 plays a pivotal role in the maintenance of stemness and therapeutic resistance in GSCs.

Molecular diversity of diencephalic astrocytes reveals adult astrogenesis regulated by Smad4

Abstract: Astrocytes regulate brain-wide functions and also show region-specific differences, but little is known about how general and region-specific functions are aligned at the single-cell level. To explore this, we isolated adult mouse diencephalic astrocytes by ACSA-2-mediated magnetic-activated cell sorting (MACS). Single-cell RNA-seq revealed 7 gene expression clusters of astrocytes, with 4 forming a supercluster. Within the supercluster, cells differed by gene expression related to ion homeostasis or metabolism, with the former sharing gene expression with other regions and the latter being restricted to specific regions. All clusters showed expression of proliferation-related genes, and proliferation of diencephalic astrocytes was confirmed by immunostaining. Clonal analysis demonstrated low level of astrogenesis in the adult diencephalon, but not in cerebral cortex grey matter. This led to the identification of Smad4 as a key regulator of diencephalic astrocyte in vivo proliferation and in vitro neurosphere formation. Thus, astrocytes show diverse gene expression states related to distinct functions with some subsets being more widespread while others are more regionally restricted. However, all share low-level proliferation revealing the novel concept of adult astrogenesis in the diencephalon.

A Phase II and Pharmacodynamic Trial of RO4929097 for Patients With Recurrent/Progressive Glioblastoma

Abstract: Background Cancer stem-like cells are a major cause of resistance to therapy in patients with glioblastoma (GBM) as well as other cancers. Tumor cells are maintained in a stem-like proliferative state in large part through the Notch signaling pathway. The function of this pathway in turn depends on gamma secretase activity. Inhibition of this enzyme therefore inhibits the Notch pathway and tumor growth as measured by a reduction in the formation of brain tumor neurospheres in murine models. RO4929097 is an oral gamma secretase inhibitor. Objective To estimate the 6-mo progression-free survival rate (PFS6) in patients with progressive GBM and to inhibit by 50% the generation of neurospheres in fresh tissue resected from patients treated with RO4929097. Methods In this phase II and pharmacodynamic study, patients with recurrent GBM received RO4929097 in a study of 2 groups. Group A patients had unresectable disease and received drug in a standard phase II design. Group B patients had resectable disease and received drug before and after surgical resection. Endpoints included PFS6 and the inhibition of neurosphere formation in the resected tumor samples. Results A total of 47 patients received treatment, 7 of whom had tumor resection. The PFS6 was 4%, and the inhibition of neurosphere formation occurred in 1 of 7 patient samples. Conclusion RO4929097 was inactive in recurrent GBM patients and demonstrated minimal inhibition of neurosphere formation in fresh tissue samples.

Effect of Long-Term 3D Spheroid Culture on WJ-MSC

Abstract: The aim of our work was to develop a protocol enabling a derivation of mesenchymal stem/stromal cell (MSC) subpopulation with increased expression of pluripotent and neural genes. For this purpose we used a 3D spheroid culture system optimal for neural stem cells propagation. Although 2D culture conditions are typical and characteristic for MSC, under special treatment these cells can be cultured for a short time in 3D conditions. We examined the effects of prolonged 3D spheroid culture on MSC in hope to select cells with primitive features. Wharton Jelly derived MSC (WJ-MSC) were cultured in 3D neurosphere induction medium for about 20 days in vitro. Then, cells were transported to 2D conditions and confront to the initial population and population constantly cultured in 2D. 3D spheroids culture of WJ-MSC resulted in increased senescence, decreased stemness and proliferation. However long-termed 3D spheroid culture allowed for selection of cells exhibiting increased expression of early neural and SSEA4 markers what might indicate the survival of cell subpopulation with unique features.

Human Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Produce Distinct Neural 3D In Vitro Models Depending on Alginate/Gellan Gum/Laminin Hydrogel Blend Properties.

Abstract: Stable and predictive neural cell culture models are a necessary premise for many research fields. However, conventional 2D models lack 3D cell-material/-cell interactions and hence do not reflect the complexity of the in vivo situation properly. Here two alginate/gellan gum/laminin (ALG/GG/LAM) hydrogel blends are presented for the fabrication of human induced pluripotent stem cell (hiPSC)-based 3D neural models. For hydrogel embedding, hiPSC-derived neural progenitor cells (hiNPCs) are used either directly or after 3D neural pre-differentiation. It is shown that stiffness and stress relaxation of the gel blends, as well as the cell differentiation strategy influence 3D model development. The embedded hiNPCs differentiate into neurons and astrocytes within the gel blends and display spontaneous intracellular calcium signals. Two fit-for-purpose models valuable for i) applications requiring a high degree of complexity, but less throughput, such as disease modeling and long-term exposure studies and ii) higher throughput applications, such as acute exposures or substance screenings are proposed. Due to their wide range of applications, adjustability, and printing capabilities, the ALG/GG/LAM based 3D neural models are of great potential for 3D neural modeling in the future.

Conditional reprogramming culture conditions facilitate growth of lower-grade glioma models.

Abstract: Background The conditional reprogramming cell culture method was developed to facilitate growth of senescence-prone normal and neoplastic epithelial cells, and involves co-culture with irradiated fibroblasts and the addition of a small molecule Rho kinase (ROCK) inhibitor. The aim of this study was to determine whether this approach would facilitate the culture of compact low grade gliomas. Methods We attempted to culture 4 pilocytic astrocytomas, 2 gangliogliomas, 2 myxopapillary ependymomas, 2 anaplastic gliomas, 2 difficult-to-classify low grade neuroepithelial tumors, a desmoplastic infantile ganglioglioma, and an anaplastic pleomorphic xanthoastrocytoma using a modified conditional reprogramming cell culture approach. Results Conditional reprogramming resulted in robust increases in growth for a majority of these tumors, with fibroblast conditioned media and ROCK inhibition both required. Switching cultures to standard serum containing media, or serum free neurosphere conditions, with or without ROCK inhibition, resulted in decreased proliferation and induction of senescence markers. ROCK inhibition and conditioned media both promoted Akt and Erk1/2 activation. Several cultures, including one derived from a NF1-associated pilocytic astrocytoma (JHH-NF1-PA1) and one from a BRAF p.V600E mutant anaplastic pleomorphic xanthoastrocytoma (JHH-PXA1), exhibited growth sufficient for preclinical testing in vitro. In addition, JHH-NF1-PA1 cells survived and migrated in larval zebrafish orthotopic xenografts, while JHH-PXA1 formed orthotopic xenografts in mice histopathologically similar to the tumor from which it was derived. Conclusions These studies highlight the potential for the conditional reprogramming cell culture method to promote the growth of glial and glioneuronal tumors in vitro, in some cases enabling the establishment of long-term culture and in vivo models.

Design and Evaluation of an In Vitro Mild Traumatic Brain Injury Modeling System Using 3D Printed Mini Impact Device on the 3D Cultured Human iPSC Derived Neural Progenitor Cells

Abstract: Despite significant progress in understanding the disease mechanism of traumatic brain injury (TBI), promising preclinical therapeutics have seldom been translated into successful clinical outcomes, partially because the model animals have physiological and functional differences in the central nervous system (CNS) compared to humans. Human relevant models are thus urgently required. Here, an in vitro mild TBI (mTBI) modeling system is reported based on 3D cultured human induced pluripotent stem cells (iPSC) derived neural progenitor cells (iPSC-NPCs) to evaluate consequences of single and repetitive mTBI using a 3D printed mini weight-drop impact device. Computational simulation is performed to understand the single/cumulative effects of weight-drop impact on the NPC differentiated neurospheres. Experimental results reveal that neurospheres show reactive astrogliosis and glial scar formation after repetitive (10 hits) mild impacts, while no astrocyte activation is found after one or two mild impacts. A 3D co-culture model of human microglia cells with neurospheres is further developed. It is found that astrocyte response is promoted even after two mild impacts, possibly caused by the chronic neuroinflammation after microglia activation. The in vitro mTBI modeling system recapitulates several hallmarks of the brain impact injury and might serve as a good platform for future drug screening.

Bioabsorbable nerve conduits three-dimensionally coated with human induced pluripotent stem cell-derived neural stem/progenitor cells promote peripheral nerve regeneration in rats

Abstract: Peripheral nerve regeneration using nerve conduits has been less effective than autogenous nerve grafts. To overcome this hurdle, we developed a tissue-engineered nerve conduit coated with mouse induced pluripotent stem cell (iPSC)-derived neurospheres, for the first time, which accelerated nerve regeneration in mice. We previously demonstrated the long-term efficacy and safety outcomes of this hybrid nerve conduit for mouse peripheral nerve regeneration. In this study, we investigated the therapeutic potential of nerve conduits coated with human iPSC (hiPSC)-derived neurospheres in rat sciatic nerve defects, as a translational preclinical study. The hiPSC-derived quaternary neurospheres containing neural stem/progenitor cells were three-dimensionally cultured within the nerve conduit (poly l-lactide and polycaprolactone copolymer) for 14 days. Complete 5-mm defects were created as a small size peripheral nerve defect in sciatic nerves of athymic nude rats and reconstructed with nerve conduit alone (control group), nerve conduits coated with hiPSC-derived neurospheres (iPS group), and autogenous nerve grafts (autograft group) (n = 8 per group). The survival of the iPSC-derived neurospheres was continuously tracked using in vivo imaging. At 12 weeks postoperatively, motor and sensory function and histological nerve regeneration were evaluated. Before implantation, the hiPSC-derived quaternary neurospheres that three-dimensional coated the nerve conduit were differentiated into Schwann-like cells. The transplanted hiPSC-derived neurospheres survived for at least 56 days after implantation. The iPS group showed non-significance higher sensory regeneration than the autograft group. Although there was no actual motor functional nerve regeneration in the three groups: control, iPS, and autograft groups, the motor function in the iPS group recovered significantly better than that in the control group, but it did not recover to the same level as that in the autograft group. Histologically, the iPS group demonstrated significantly higher axon numbers and areas, and lower G-ratio values than the control group, whereas the autograft group demonstrated the highest axon numbers and areas and the lowest G-ratio values. Nerve conduit three-dimensionally coated with hiPSC-derived neurospheres promoted axonal regeneration and functional recovery in repairing rat sciatic nerve small size defects. Transplantation of hiPSC-derived neurospheres with nerve conduits is a promising clinical iPSC-based cell therapy for the treatment of peripheral nerve defects.

Neurospheres: a potential in vitro model for the study of central nervous system disorders

Abstract: Neurogenesis was believed to end after the period of embryonic development. However, the possibility of obtaining an expressive number of cells with functional neuronal characteristics implied a great advance in experimental research. New techniques have emerged to demonstrate that the birth of new neurons continues to occur in the adult brain. Two main rich sources of these cells are the subventricular zone (SVZ) and the subgranular zone of the hippocampal dentate gyrus (SGZ) where adult neural stem cells (aNSCs) have the ability to proliferate and differentiate into mature cell lines. The cultivation of neurospheres is a method to isolate, maintain and expand neural stem cells (NSCs) and has been used extensively by several research groups to analyze the biological properties of NSCs and their potential use in injured brains from animal models. Throughout this review, we highlight the areas where this type of cell culture has been applied and the advantages and limitations of using this model in experimental studies for the neurological clinical scenario.

Inhibition of Gli2 suppresses tumorigenicity in glioblastoma stem cells derived from a de novo murine brain cancer model

Abstract: The prognosis of glioblastoma remains poor despite intensive research efforts. Glioblastoma stem cells (GSCs) contribute to tumorigenesis, invasive capacity, and therapy resistance. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), a stem cell marker, is involved in the maintenance of GSCs, although the properties of Lgr5-positive GSCs remain unclear. Here, the Sleeping-Beauty transposon-induced glioblastoma model was used in Lgr5-GFP knock-in mice identify GFP-positive cells in neurosphere cultures from mouse glioblastoma tissues. Global gene expression analysis showed that Gli2 was highly expressed in GFP-positive GSCs. Gli2 knockdown using lentiviral-mediated shRNA downregulated Hedgehog-related and Wnt signaling pathway-related genes, including Lgr5; suppressed tumor cell proliferation and invasion capacity; and induced apoptosis. Pharmacological Gli inhibition with GANT61 suppressed tumor cell proliferation. Silencing Gli2 suppressed the tumorigenicity of GSCs in an orthotopic transplantation model in vivo. These findings suggest that Gli2 affects the Hedgehog and Wnt pathways and plays an important role in GSC maintenance, suggesting Gli2 as a therapeutic target for glioblastoma treatment.

Enteric mesenchymal cells support the growth of postnatal enteric neural stem cells.

Abstract: Interplay between embryonic enteric neural stem cells (ENSCs) and enteric mesenchymal cells (EMCs) in the embryonic gut is essential for normal development of the enteric nervous system. Disruption of these interactions underlies the pathogenesis of intestinal aganglionosis in Hirschsprung disease (HSCR). ENSC therapy has been proposed as a possible treatment for HSCR, but whether the survival and development of postnatal-derived ENSCs similarly rely on signals from the mesenchymal environment is unknown and has important implications for developing protocols to expand ENSCs for cell transplantation therapy. Enteric neural crest-derived cells (ENCDCs) and EMCs were cultured from the small intestine of Wnt1-Rosa26-tdTomato mice. EMCs promoted the expansion of ENCDCs 9.5-fold by inducing ENSC properties, including expression of Nes, Sox10, Sox2, and Ngfr. EMCs enhanced the neurosphere-forming ability of ENCDCs, and this persisted after withdrawal of the EMCs. These effects were mediated by paracrine factors and several ligands known to support neural stem cells were identified in EMCs. Using the optimized expansion procedures, neurospheres were generated from small intestine of the Ednrb-/- mouse model of HSCR. These ENSCs had similar proliferative and migratory capacity to Ednrb+/+ ENSCs, albeit neurospheres contained fewer neurons. ENSCs derived from Ednrb-/- mice generated functional neurons with similar calcium responses to Ednrb+/+ ENSCs and survived after transplantation into the aganglionic colon of Ednrb-/- recipients. EMCs act as supporting cells to ENSCs postnatally via an array of synergistically acting paracrine signaling factors. These properties can be leveraged to expand autologous ENSCs from patients with HSCR mutations for therapeutic application.

Neural stemness contributes to cell tumorigenicity.

Abstract: Previous studies demonstrated the dependence of cancer on nerve. Recently, a growing number of studies reveal that cancer cells share the property and regulatory network with neural stem/progenitor cells. However, relationship between the property of neural stemness and cell tumorigenicity is unknown. We show that neural stem/progenitor cells, but not non-neural embryonic or somatic stem/progenitor cell types, exhibit tumorigenicity and the potential for differentiation into tissue types of all germ layers when they are placed in non-native environment by transplantation into immunodeficient nude mice. Likewise, cancer cells capable of tumor initiation have the property of neural stemness because of their abilities in neurosphere formation in neural stem cell-specific serum-free medium and in differentiation potential, in addition to their neuronal differentiation potential that was characterized previously. Moreover, loss of a pro-differentiation factor in myoblasts, which have no tumorigenicity, lead to the loss of myoblast identity, and gain of the property of neural stemness, tumorigenicity and potential for re-differentiation. By contrast, loss of neural stemness via differentiation results in the loss of tumorigenicity. These suggest that the property of neural stemness contributes to cell tumorigenicity, and tumor phenotypic heterogeneity might be an effect of differentiation potential of neural stemness. Bioinformatic analysis reveals that neural genes in general are correlated with embryonic development and cancer, in addition to their role in neural development; whereas non-neural genes are not. Most of neural specific genes emerged in typical species representing transition from unicellularity to multicellularity during evolution. Genes in Monosiga brevicollis, a unicellular species that is a closest known relative of metazoans, are biased toward neural cells. We suggest that the property of neural stemness is the source of cell tumorigenicity. This is due to that neural biased unicellular state is the ground state for multicellularity and hence cell type diversification or differentiation during evolution, and tumorigenesis is a process of restoration of neural ground state in somatic cells along a default route that is pre-determined by an evolutionary advantage of neural state.

Microalgae Aurantiochytrium Sp. Increases Neurogenesis and Improves Spatial Learning and Memory in Senescence-Accelerated Mouse-Prone 8 Mice.

Abstract: Much attention has recently been focused on nutraceuticals, with minimal adverse effects, developed for preventing or treating neurological diseases such as Alzheimer's disease (AD) The present study was conducted to investigate the potential effect on neural development and function of the microalgae Aurantiochytrium sp as a nutraceutical To test neuroprotection by the ethanol extract of Aurantiochytrium (EEA) and a derivative, the n-Hexane layer of EEA (HEEA), amyloid-β-stimulated SH-SY5Y cells, was used as an in vitro AD model We then assessed the potential enhancement of neurogenesis by EEA and HEEA using murine ex vivo neurospheres We also administered EEA or HEEA to senescence-accelerated mouse-prone 8 (SAMP8) mice, a non-transgenic strain with accelerated aging and AD-like memory loss for evaluation of spatial learning and memory using the Morris water maze test Finally, we performed immunohistochemical analysis for assessment of neurogenesis in mice administered EEA Pretreatment of SH-SY5Y cells with EEA or the squalene-rich fraction of EEA, HEEA, ameliorated amyloid-β-induced cytotoxicity Interestingly, only EEA-treated cells showed a significant increase in cell metabolism and intracellular adenosine triphosphate production Moreover, EEA treatment significantly increased the number of neurospheres, whereas HEEA treatment significantly increased the number of β-III-tubulin+ young neurons and GFAP+ astrocytes SAMP8 mice were given 50 mg/kg EEA or HEEA orally for 30 days EEA and HEEA decreased escape latency in the Morris water maze in SAMP8 mice, indicating improved memory To detect stem cells and newborn neurons, we administered BrdU for 9 days and measured BrdU+ cells in the dentate gyrus, a neurogenic stem cell niche of the hippocampus In SAMP8 mice, EEA rapidly and significantly increased the number of BrdU+GFAP+ stem cells and their progeny, BrdU+NeuN+ mature neurons In conclusion, our data in aggregate indicate that EEA and its constituents could be developed into a nutraceutical for promoting brain health and function against several age-related diseases, particularly AD

Rabbit neurospheres as a novel in vitro tool for studying neurodevelopmental effects induced by intrauterine growth restriction.

Abstract: The aim of this study was to develop a rabbit neurosphere culture to characterize differences in basic processes of neurogenesis induced by intrauterine growth restriction (IUGR). A novel in vitro neurosphere culture has been established using fresh or frozen neural progenitor cells from newborn (PND0) rabbit brains. After surgical IUGR induction in pregnant rabbits and cesarean section 5 days later, neural progenitor cells from both control and IUGR groups were isolated and directly cultured or frozen at -80°C. These neural progenitor cells spontaneously formed neurospheres after 7 days in culture. The ability of control and IUGR neurospheres to migrate, proliferate, differentiate to neurons, astrocytes, or oligodendrocytes was compared and the possibility to modulate their responses was tested by exposure to several positive and negative controls. Neurospheres obtained from IUGR brains have a significant impairment in oligodendrocyte differentiation, whereas no significant differences are observed in other basic processes of neurogenesis. This impairment can be reverted by in vitro exposure of IUGR neurospheres to thyroid hormone, which is known to play an essential role in white matter maturation in vivo. Our new rabbit neurosphere model and the results of this study open the possibility to test several substances in vitro as neuroprotective candidates against IUGR induced neurodevelopmental damage while decreasing the number of animals and resources and allowing a more mechanistic approach at a cellular functional level.

Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro.

Abstract: The generation of mature synaptic structures using neurons differentiated from human-induced pluripotent stem cells (hiPSC-neurons) is expected to be applied to physiological studies of synapses in human cells and to pathological studies of diseases that cause abnormal synaptic function. Although it has been reported that synapses themselves change from an immature to a mature state as neurons mature, there are few reports that clearly show when and how human stem cell-derived neurons change to mature synaptic structures. This study was designed to elucidate the synapse formation process of hiPSC-neurons. We propagated hiPSC-derived neural progenitor cells (hiPSC-NPCs) that expressed localized markers of the ventral hindbrain as neurospheres by dual SMAD inhibition and then differentiated them into hiPSC-neurons in vitro. After 49 days of in vitro differentiation, hiPSC-neurons significantly expressed pre- and postsynaptic markers at both the transcript and protein levels. However, the expression of postsynaptic markers was lower than in normal human or normal rat brain tissues, and immunostaining analysis showed that it was relatively modest and was lower than that of presynaptic markers and that its localization in synaptic structures was insufficient. Neurophysiological analysis using a microelectrode array also revealed that no synaptic activity was generated on hiPSC-neurons at 49 days of differentiation. Analysis of subtype markers by immunostaining revealed that most hiPSC-neurons expressed vesicular glutamate transporter 2 (VGLUT2). The presence or absence of NGF, which is required for the survival of cholinergic neurons, had no effect on their cell fractionation. These results suggest that during the synaptogenesis of hiPSC-neurons, the formation of presynaptic structures is not the only requirement for the formation of postsynaptic structures and that the mRNA expression of postsynaptic markers does not correlate with the formation of their mature structures. Technically, we also confirmed a certain level of robustness and reproducibility of our neuronal differentiation method in a multicenter setting, which will be helpful for future research. Synapse formation with mature postsynaptic structures will remain an interesting issue for stem cell-derived neurons, and the present method can be used to obtain early and stable quality neuronal cultures from hiPSC-NPCs.

Molecular genetic analysis of neural stem cells after space flight and simulated microgravity on earth.

Abstract: Understanding how stem cells adapt to space flight conditions is fundamental for human space missions and extraterrestrial settlement. We analyzed gene expression in boundary cap neural crest stem cells (BC), which are attractive for regenerative medicine by their ability to promote proliferation and survival of co-cultured and co-implanted cells. BC were launched to space (space exposed cells) (SEC), on board sounding rocket MASER 14 as free-floating neurospheres or in bioprinted scaffold. For comparison, BC were placed in a random positioning machine (RPM) to simulate microgravity on earth (RPM cells) or were cultured under control conditions in the laboratory. Using Next-Generation RNA sequencing and data post-processing, we discovered that SEC upregulated genes related to proliferation and survival, whereas RPM cells upregulated genes associated with differentiation and inflammation. Thus, i) space flight provides unique conditions with distinctly different effects on the properties of BC compared to earth controls, and ii) the space flight exposure induces post-flight properties that reinforce the utility of BC for regenerative medicine and tissue engineering. This article is protected by copyright. All rights reserved.

Morphological and biochemical repercussions of Toxoplasma gondii infection in a 3D human brain neurospheres model.

Abstract: Background Toxoplasmosis is caused by the parasite Toxoplasma gondii that can infect the central nervous system (CNS), promoting neuroinflammation, neuronal loss, neurotransmitter imbalance and behavioral alterations. T. gondii infection is also related to neuropsychiatric disorders such as schizophrenia. The pathogenicity and inflammatory response in rodents are different to the case of humans, compromising the correlation between the behavioral alterations and physiological modifications observed in the disease. In the present work we used BrainSpheres, a 3D CNS model derived from human pluripotent stem cells (iPSC), to investigate the morphological and biochemical repercussions of T. gondii infection in human neural cells. Methods We evaluated T. gondii ME49 strain proliferation and cyst formation in both 2D cultured human neural cells and BrainSpheres. Aspects of cell morphology, ultrastructure, viability, gene expression of neural phenotype markers, as well as secretion of inflammatory mediators were evaluated for 2 and 4 weeks post infection in BrainSpheres. Results T. gondii can infect BrainSpheres, proliferating and inducing cysts formation, neural cell death, alteration in neural gene expression and triggering the release of several inflammatory mediators. Conclusions BrainSpheres reproduce many aspects of T. gondii infection in human CNS, constituting a useful model to study the neurotoxicity and neuroinflammation mediated by the parasite. In addition, these data could be important for future studies aiming at better understanding possible correlations between psychiatric disorders and human CNS infection with T. gondii.

RNA Profiling of Mouse Ependymal Cells after Spinal Cord Injury Identifies the Oncostatin Pathway as a Potential Key Regulator of Spinal Cord Stem Cell Fate

Abstract: Ependymal cells reside in the adult spinal cord and display stem cell properties in vitro. They proliferate after spinal cord injury and produce neurons in lower vertebrates but predominantly astrocytes in mammals. The mechanisms underlying this glial-biased differentiation remain ill-defined. We addressed this issue by generating a molecular resource through RNA profiling of ependymal cells before and after injury. We found that these cells activate STAT3 and ERK/MAPK signaling post injury and downregulate cilia-associated genes and FOXJ1, a central transcription factor in ciliogenesis. Conversely, they upregulate 510 genes, seven of them more than 20-fold, namely Crym, Ecm1, Ifi202b, Nupr1, Rbp1, Thbs2 and Osmr—the receptor for oncostatin, a microglia-specific cytokine which too is strongly upregulated after injury. We studied the regulation and role of Osmr using neurospheres derived from the adult spinal cord. We found that oncostatin induced strong Osmr and p-STAT3 expression in these cells which is associated with reduction of proliferation and promotion of astrocytic versus oligodendrocytic differentiation. Microglial cells are apposed to ependymal cells in vivo and co-culture experiments showed that these cells upregulate Osmr in neurosphere cultures. Collectively, these results support the notion that microglial cells and Osmr/Oncostatin pathway may regulate the astrocytic fate of ependymal cells in spinal cord injury.

Natural Membrane Differentiates Human Adipose-Derived Mesenchymal Stem Cells to Neurospheres by Mechanotransduction Related to YAP and AMOT Proteins.

Abstract: Adipose tissue-derived mesenchymal stem cells (ADMSCs) are promising candidates for regenerative medicine, as they have good cell yield and can differentiate into several cell lines. When induced to the neuronal differentiation, they form neurospheres composed of neural precursors (NPs) that can be an alternative in treating neurodegenerative diseases. This study aimed to characterize NPs from neurospheres obtained after seeding ADMSCs on a natural polyisoprene-based membrane. The ADMSCs were isolated from adipose tissue by enzymatic dissociation, were subjected to trilineage differentiation, and were characterized by flow cytometry for specific ADMSC surface markers. For neuronal differentiation, the cells were seeded on polystyrene flasks coated with the membrane and were characterized by immunocytochemistry and RT-PCR. The results demonstrated that the isolated cells showed characteristics of ADMSCs. At 15 to 25 days, ADMSCs seeded on the natural membrane developed neurospheres. Then, after dissociation, the cells demonstrated characteristic neuronal markers expressed on NPs: nestin, s-III tubulin, GFAP, NeuN, and the YAP1/AMOT in the cytoplasm. In conclusion, it was demonstrated that this membrane differentiates the ADMSCs to NPs without any induction factors, and suggests that their differentiation mechanisms are related to mechanotransduction regulated by the YAP and AMOT proteins.

PIM1 Inhibition Affects Glioblastoma Stem Cell Behavior and Kills Glioblastoma Stem-like Cells.

Abstract: Despite comprehensive therapy and extensive research, glioblastoma (GBM) still represents the most aggressive brain tumor in adults. Glioma stem cells (GSCs) are thought to play a major role in tumor progression and resistance of GBM cells to radiochemotherapy. The PIM1 kinase has become a focus in cancer research. We have previously demonstrated that PIM1 is involved in survival of GBM cells and in GBM growth in a mouse model. However, little is known about the importance of PIM1 in cancer stem cells. Here, we report on the role of PIM1 in GBM stem cell behavior and killing. PIM1 inhibition negatively regulates the protein expression of the stem cell markers CD133 and Nestin in GBM cells (LN-18, U-87 MG). In contrast, CD44 and the astrocytic differentiation marker GFAP were up-regulated. Furthermore, PIM1 expression was increased in neurospheres as a model of GBM stem-like cells. Treatment of neurospheres with PIM1 inhibitors (TCS PIM1-1, Quercetagetin, and LY294002) diminished the cell viability associated with reduced DNA synthesis rate, increased caspase 3 activity, decreased PCNA protein expression, and reduced neurosphere formation. Our results indicate that PIM1 affects the glioblastoma stem cell behavior, and its inhibition kills glioblastoma stem-like cells, pointing to PIM1 targeting as a potential anti-glioblastoma therapy.

Functionalized Graphene Quantum Dots Modulate Malignancy of Glioblastoma Multiforme by Downregulating Neurospheres Formation

Abstract: Glioblastoma multiforme (GBM) is the most aggressive brain cancer. We previously demonstrated the effect of biocompatible surface functionalized graphene quantum dots (GQDs) on GBM cells as chemotherapy enhancers in combination with the antitumor drug doxorubicin (Dox). However, traditional two-dimensional cultures could not represent a reliable model of tumor behavior. In this work, we investigated the effect of carboxylated (COOH-GQDs), aminated (NH2-GQDs) and unfunctionalized GQDs on a three-dimensional model of neurospheres. Neurospheres are clusters of GBM cells, which formation is driven by the presence of a stem subpopulation involved in cancer malignancy. Tumor recurrence after surgical resection, chemotherapy and radiotherapy indeed depends on the presence of cancer cells with stem properties. We measured a significant reduction in number and size of neurospheres after two weeks of monitoring in the presence of COOH-GQDs and GQDs. Previous works pointed out how variations of membrane fluidity could affect membrane stability and cell-to-cell interactions, thus influencing cell clustering. Therefore, we measured changes in membrane fluidity after administration of GQDs. We found that COOH-GQDs and GQDs significantly increased membrane fluidity with respect to the treatment with NH2-GQDs or compared to untreated cells. Shifts in the phase of phospholipid bilayer were in accordance with the negative surface net charge of GQDs. We depicted a strong correlation between negatively charged GQDs-induced increase in membrane fluidity and the downregulation of neurospheres formation. Our results indicate that COOH-GQDs and GQDs significantly modulate tumor malignancy by increasing fluidity of cell membrane, with a consequent inhibition of cell-to-cell interaction.

Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept.

Abstract: Failure of corneal endothelium cell monolayer is the main cause leading to corneal transplantation Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer Several strategies have been proposed using embryonic stem cells and induced pluripotent stem cells, although their use has ethical issues as well as limited clinical applications For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form the corneal endothelium cell monolayer We hypothesize that using dental pulp stem cells that share an embryological origin with corneal endothelial cells, as they both arise from the neural crest, may allow a direct differentiation process avoiding the use of reprogramming techniques, such as induced pluripotent stem cells In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem-like cells and, then, into corneal endothelial-like cells Initially, for the first-step we used an adhesion culture and compared two initial cell sources: a direct formation from dental pulp stem cells with the differentiation from induced pluripotent stem cells Results showed significantly higher levels of early stage marker AP2 for the dental pulp stem cells compared to induced pluripotent stem cells In order to provide a better environment for neural crest stem cells generation, we performed a suspension method, which induced the formation of neurospheres Results showed that neurosphere formation obtained the peak of neural crest stem cell markers expression after 4 days, showing overexpression of AP2, Nestin, and p75 markers, confirming the formation of neural crest stem-like cells Furthermore, pluripotent markers Oct4, Nanog, and Sox2 were as well-upregulated in suspension culture Neurospheres were then directly cultured in corneal endothelial conditioned medium for the second differentiation into corneal endothelial-like cells Results showed the conversion of dental pulp stem cells into polygonal-like cells expressing higher levels of ZO-1, ATP1A1, COL4A2, and COL8A2 markers, providing a proof of the conversion into corneal endothelial-like cells Therefore, our findings demonstrate that patient-derived dental pulp stem cells may represent an autologous cell source for corneal endothelial therapies that avoids actual transplantation limitations as well as reprogramming techniques