Formation Of Neural Stem Cells Research Articles

Early neural specification of stem cells is mediated by a set of SOX2-dependent neural-associated enhancers

SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In addition, SOX2 plays a pivotal role in neural stem cell formation and neurogenesis. How SOX2 can serve both processes has remained elusive. Here, we identified a set of SOX2-dependent neural-associated enhancers required for neural lineage priming. They form a distinct subgroup (1,898) among 8,531 OCT4/SOX2/NANOG-bound enhancers characterized by enhanced SOX2 binding and chromatin accessibility. Activation of these enhancers is triggered by neural induction of wild-type cells or by default in Smad4-ablated cells resistant to mesoderm induction and is antagonized by mesodermal transcription factors via Sox2 repression. Our data provide mechanistic insight into the transition from the pluripotency state to the early neural fate and into the regulation of early neural versus mesodermal specification in embryonic stem cells and embryos.

Abstract 3100: The development of pleotropic small molecule kinase inhibitors (SKMIs) for the treatment of glioblastoma multiforme (GBM)

Abstract Glioblastoma multiforme (GBM) is an aggressive stage IV brain cancer that is difficult to detect, typically inoperable, and associated with a poor prognosis. Challenges in targeting GBM arise from the involvement of various pro-survival pathways, being comprised of four different cellular states, and displaying heterogeneity between each tumor. Marketed FDA-approved drugs for GBM have demonstrated limited efficacy, toxicity issues, resistance, and poor BBB penetrance. Further probing into pathway breakdowns led to the discovery of the DYRKs (dual-specificity tyrosine-regulated kinase), CLKs (CDC-like kinases), PDGFRA/B (platelet-derived growth factor receptor), and PI3Kα (phosphoinositide 3-kinase) involvement and overexpression. The upregulation of these kinases are found to be prominently involved in GBM cell proliferation, survival, replication, aggression, metastasis, and resistance. Herein, we believe that the development of pleiotropic competitive inhibitors for the treatment of GBM could be a promising therapeutic option based on literature precedence and current data from research. DYR726, a water-soluble, pleiotropic inhibitor [IC50 PDGFRA/B/PI3Kα/CLk2/3DYRK1a/2/3 ( 53.0/48.0/6.2/15.13/137/4/38.6/11.2 nM)] has demonstrated in vitro cellular efficacy across various GBM cell lines. When treated in non-cancerous cell lines, DYR726 displayed an optimal therapeutic window, which is not observed in any glioma drugs currently in clinical trials. At 1µM concentrations, complete dissociation of the formation of neural stem cells and neurosphere growth in glioma cells are observed. Reduction in metastasis and proliferation are found when treated with DYR726 over three weeks. In vitro efficacy against current approved FDA treatment options and clinical candidates has been extensively tested across numerous GBM cell lines. When benched against one of the best PI3K kinase inhibitors in the clinic, Buparlisib, DYR726 was comparable, indicating a disruption in the RTK signaling survival pathway that is commonly associated with GBM. DYR726 performed favorably against Avapritinib (clinical trials for gliomas are beginning at Stanford) and when treated against FDA-approved combination treatment Dabrafenib+Trametinib (for 2% of glioma patients), it performed identically, suggesting that DYR726 can inhibit the cells at the same rate as the new FDA drug. Within patient-derived cell lines, it behaves identically to Buparlisib, and DYR726 has been shown to outperform AstraZenecas’ Osimertinib (which was significantly less potent) and Tafinlars’ Dabrafenib (displayed no effect). Based on the significance of efficacy, this series is on the CRUK Glasgow Cancer Center grant renewal as a potential Phase 0 within the next five years. Citation Format: Alessandra Fistrovich, Vasudah Tandon, Carly Cabel, Laura Basantes, Curtis Thorne, William Montfort, Sourav Banerjee, Christopher Hulme. The development of pleotropic small molecule kinase inhibitors (SKMIs) for the treatment of glioblastoma multiforme (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3100.

The cortical hem lacks stem cell potential despite expressing SOX9 and HOPX.

The adult dentate gyrus (DG) of rodents hosts a neural stem cell (NSC) niche capable of generating new neurons throughout life. The embryonic origin and molecular mechanisms underlying formation of DG NSCs are still being investigated. We performed a bulk transcriptomic analysis on mouse developing archicortex conditionally deleted for Sox9, a SoxE transcription factor controlling both gliogenesis and NSC formation, and identified Hopx, a recently identified marker of both prospective adult DG NSCs and astrocytic progenitors, as being downregulated. We confirm SOX9 is required for HOPX expression in the embryonic archicortex. In particular, we found that both NSC markers are highly expressed in the cortical hem (CH), while only weakly in the adjacent dentate neuroepithelium (DNE), suggesting a potential CH embryonic origin for DG NSCs. However, we demonstrate both in vitro and in vivo that the embryonic CH, as well as its adult derivatives, lacks stem cell potential. Instead, deletion of Sox9 in the DNE affects both HOPX expression and NSC formation in the adult DG. We conclude that HOPX expression in the CH is involved in astrocytic differentiation downstream of SOX9, which we previously showed regulates DG development by inducing formation of a CH-derived astrocytic scaffold. Altogether, these results suggest that both proteins work in a dose-dependent manner to drive either astrocytic differentiation in CH or NSC formation in DNE.

Upscaling biological complexity to boost neuronal and oligodendroglia maturation and improve in vitro developmental neurotoxicity (DNT) evaluation

Human induced pluripotent stem cell (iPSC)-derived neuronal and glial cell models are suitable to assess the effects of environmental chemicals on the developing brain. Such test systems can recapitulate several key neurodevelopmental features, such as neural stem cell formation and differentiation towards different neuronal subtypes and astrocytes, neurite outgrowth, synapse formation and neuronal network formation and function, which are crucial for brain development. While monolayer, two-dimensional (2D) cultures of human iPSC-neuronal or glial derivatives are generally suited for high-throughput testing, they also show some limitations. In particular, differentiation towards myelinating oligodendrocytes can only be achieved after extended periods in differentiation. In recent years, the implementation of three-dimensional (3D) neuronal and glial models obtained from human iPSCs has been shown to compensate for such limitations, enabling robust differentiation towards both neuronal and glial cell populations, myelination and formation of more mature neuronal network activity. Here we compared the differentiation capacity of human iPSC-derived neural stem cells cultured either as 2D monolayer or as 3D neurospheres, and assessed chlorpyrifos (CPF) effects. Data indicate that 3D neurospheres differentiate towards neurons and oligodendroglia more rapidly than 2D cultures; however, the 2D model is more suitable to assess neuronal functionality by analysis of spontaneous electrical activity using multielectrode array. Moreover, 2D and 3D test systems are diversely susceptible to CPF treatment. In conclusion, the selection of the most suitable in vitro test system (either 2D or 3D) should take into account the context of use and intended research goals (‘fit for purpose’ principle).

MiR-1297 regulates neural stem cell differentiation and viability through controlling Hes1 expression.

Neural stem cells (NSCs) are self-renewing, undifferentiated and multipotent precursors that can generate neuronal and glial lineages. MicroRNAs (miRNAs) are small non-coding RNAs that act crucial roles in cell proliferation, differentiation and migration. However, the role of miR-1297 in the development of NSCs is still unknown. Primary NSCs were isolated from rat's embryos. The expression of miR-1297 and Hes1 were measured by qRT-PCR. Western blot was performed to detect the protein expression of Hes1, β-tubulin-III and GFAP. We showed that miR-1297 expression was upregulated during NSC differentiation, while the expression of Hes1 was decreased during NSC differentiation. Elevated expression of miR-1297 promoted the NSCs viability and increased the formation of NSCs to neurospheres. Ecoptic expression of miR-1297 promoted β-tubulin-III expression in the NSCs. Overexpression of miR-1297 decreased GFAP expression in the NSCs. Furthermore, we demonstrated that miR-1297 regulated NSCs viability and differentiation by directly targeting Hes1. Overexpression of miR-1297 suppressed Hes1 expression in the NSCs. These results suggested that miR-1297 played an important role in NSCs viability and differentiation through inhibiting Hes1 expression.

Conversion of Nonproliferating Astrocytes into Neurogenic Neural Stem Cells: Control by FGF2 and Interferon-γ.

Conversion of astrocytes to neurons, via de-differentiation to neural stem cells (NSC), may be a new approach to treat neurodegenerative diseases and brain injuries. The signaling factors affecting such a cell conversion are poorly understood, and they are hard to identify in complex disease models or conventional cell cultures. To address this question, we developed a serum-free, strictly controlled culture system of pure and homogeneous "astrocytes generated from murine embryonic stem cells (ESC)." These stem cell derived astrocytes (mAGES), as well as standard primary astrocytes resumed proliferation upon addition of FGF. The signaling of FGF receptor tyrosine kinase converted GFAP-positive mAGES to nestin-positive NSC. ERK phosphorylation was necessary, but not sufficient, for cell cycle re-entry, as EGF triggered no de-differentiation. The NSC obtained by de-differentiation of mAGES were similar to those obtained directly by differentiation of ESC, as evidenced by standard phenotyping, and also by transcriptome mapping, metabolic profiling, and by differentiation to neurons or astrocytes. The de-differentiation was negatively affected by inflammatory mediators, and in particular, interferon-γ strongly impaired the formation of NSC from mAGES by a pathway involving phosphorylation of STAT1, but not the generation of nitric oxide. Thus, two antagonistic signaling pathways were identified here that affect fate conversion of astrocytes independent of genetic manipulation. The complex interplay of the respective signaling molecules that promote/inhibit astrocyte de-differentiation may explain why astrocytes do not readily form neural stem cells in most diseases. Increased knowledge of such factors may provide therapeutic opportunities to favor such conversions. Stem Cells 2016;34:2861-2874.

Phospholipase D1 increases Bcl-2 expression during neuronal differentiation of rat neural stem cells.

We studied the possible role of phospholipase D1 (PLD1) in the neuronal differentiation, including neurite formation of neural stem cells. PLD1 protein and PLD activity increased during neuronal differentiation. Bcl-2 also increased. Downregulation of PLD1 by transfection with PLD1 siRNA or a dominant-negative form of PLD1 (DN-PLD1) inhibited both neurite outgrowth and Bcl-2 expression. PLD activity was dramatically reduced by a PLCγ (phospholipase Cγ) inhibitor (U73122), a Ca(2+)chelator (BAPTA-AM), and a PKCα (protein kinase Cα) inhibitor (RO320432). Furthermore, treatment with arachidonic acid (AA) which is generated by the action of PLA2 (phospholipase A2) on phosphatidic acid (a PLD1 product), increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, indicating that PLA2 is involved in the differentiation process resulting from PLD1 activation. PGE2 (prostaglandin E2), a cyclooxygenase product of AA, also increased during neuronal differentiation. Moreover, treatment with PGE2 increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, and this effect was inhibited by a PKA inhibitor (Rp-cAMP). As expected, inhibition of p38 MAPK resulted in loss of CREB activity, and when CREB activity was blocked with CREB siRNA, Bcl-2 production also decreased. We also showed that the EP4 receptor was required for the PKA/p38MAPK/CREB/Bcl-2 pathway. Taken together, these observations indicate that PLD1 is activated by PLCγ/PKCα signaling and stimulate Bcl-2 expression through PLA2/Cox2/EP4/PKA/p38MAPK/CREB during neuronal differentiation of rat neural stem cells.

Reconstituting Vascular Microenvironment of Neural Stem Cell Niche in Three‐Dimensional Extracellular Matrix

Neural stem cells (NSCs) reside in a vascular microenvironment termed the "NSC niche." Blood vessels in the NSC niche play an important role in maintaining an appropriate balance between NSC self-renewal and differentiation that serves to maintain homeostasis. Understanding the role of brain vessels in the NSC niche will facilitate basic research in neurogenesis and vasculogenesis as well as aid the development of potential therapies for degenerative disorders. Here, an in vitro-reconstituted NSC-vascular niche consisting of a 3D brain vasculature and extracellular matrix (ECM) microenvironment that allows NSCs to adopt physiologically relevant phenotypes through the combined effects of ECM components, chemical gradients, and signaling effectors from the brain vasculature is described. The reconstituted niche can provide precise spatiotemporal control of the NSC niche, regulating self-renewal, proliferation and colony formation of NSCs, and suppressing neuronal generation but promoting NSC differentiation into astrocytes and oligodendrocytes. It is proved that Notch effectors regulate both the astrocyte differentiation and NSC self-renewal, but the astrocyte differentiation is more active in NSCs in close proximity to brain vasculature. A potential role of the other vascular microenvironmental factor of pigment epithelium-derived factor from brain vasculature in the regulation of NSC self-renewal is also proved.

Icariin promotes self-renewal of neural stem cells: An involvement of extracellular regulated kinase signaling pathway

To investigate the effects and underlying molecular mechanisms of icariin (ICA) on self-renewal and differentiation of neural stem cells (NSCs). NSCs were derived from forebrains of mice embryos by mechanical dissociation into single cell suspension. The self-renewal of NSCs was measured by neurosphere formation assay. The proliferation of NSCs was detected by water-soluble tetrazolium (WST) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Protein expression of neuron-specific marker tubulin-βIII(TuJ1) and astrocyte-specific marker glial fibrillary acidic protein (GFAP) were measured by immunofluorescence and Western blotting. Using microarray, the differentially expressed genes (DEGs) were screened between NSCs with or without ICA treatment. The signaling pathways enriched by these DEGs and their role in mediating effects of ICA were analyzed. ICA significantly promoted neurosphere formation of NSCs cultured in growth protocol in a dose-dependent manner and achieved the maximum effects at 100 nmol/L. ICA also increased optical absorbance value and EdU incorporation into nuclei of NSCs. ICA had no significant effects on the percentage of TuJ1 or GFAP-positive cells, and TuJ1 or GFAP protein expression in NSCs cultured in differentiation protocol. A total of 478 genes were found to be differentially regulated. Among signaling pathways significantly enriched by DEGs, mitogen activated protein kinase (MAPK) pathway was of interest. Blockade of extracellular signal-regulated kinase (ERK)/MAPK, other than p38/MAPK subfamily pathway partially abolished effects of ICA on neurosphere formation and EdU incorporation of NSCs. ICA can promote the selfrenewal of NSCs at least partially through ERK/MAPK signaling pathway.

Necdin Controls Proliferation and Apoptosis of Embryonic Neural Stem Cells in an Oxygen Tension-Dependent Manner

Neural stem cells (NSCs) reside in vivo in hypoxic environments, and NSC proliferation is enhanced in vitro under hypoxic conditions. Various adaptive responses to hypoxia are mediated by hypoxia-inducible factors (HIFs), a family of basic helix-loop-helix Per-Arnt-Sim (PAS) transcription factors. Necdin, a MAGE (melanoma antigen) family protein, is expressed abundantly in postmitotic neurons and possesses potent antimitotic and antiapoptotic activities. We here report that hypoxia induces degradation of the necdin protein in primary NSCs by HIF-mediated ubiquitin-proteasome system. Necdin was expressed in primary NSCs prepared from the ganglionic eminences of mouse embryos. Hypoxia enhanced neurosphere formation of NSCs, in which the necdin protein level was significantly reduced. Primary NSCs prepared from necdin-deficient mice exhibited higher rates of proliferation and apoptosis than those from wild-type mice in normoxia, whereas there were no significant differences in the proliferation and apoptosis rates between necdin-deficient and wild-type NSCs in hypoxia. HIF-2α was predominantly expressed in hypoxic NSCs, where expression of HIF-responsive genes was upregulated. HIF-2α interacted with necdin via its PAS domain, which enhanced necdin ubiquitination. Lentivirus-mediated expression of the PAS domain in primary NSCs promoted necdin degradation and enhanced NSC proliferation in normoxia, whereas a small-molecule inhibitor of HIF-2α translation stabilized the necdin protein and reduced NSC proliferation in hypoxia. These results suggest that oxygen tension regulates the necdin protein level in NSCs through HIF-2α-mediated proteasomal degradation to modulate their proliferation and apoptosis.

Co-transplantation of neural stem cells and Schwann cells within poly (L-lactic-co-glycolic acid) scaffolds facilitates axonal regeneration in hemisected rat spinal cord

Background Various tissue engineering strategies have been developed to facilitate axonal regeneration after spinal cord injury. This study aimed to investigate whether neural stem cells (NSCs) could survive in poly(L-lactic-co-glycolic acid) (PLGA) scaffolds and, when cografted with Schwann cells (SCs), could be induced to differentiate towards neurons which form synaptic connection and eventually facilitate axonal regeneration and myelination and motor function. Methods NSCs and SCs which were seeded within the directional PLGA scaffolds were implanted in hemisected adult rat spinal cord. Control rats were similarly injured and implanted of scaffolds with or without NSCs. Survival, migration, differentiation, synaptic formation of NSCs, axonal regeneration and myelination and motor function were analyzed. Student's t test was used to determine differences in surviving percentage of NSCs. One-way analysis of variance (ANOVA) was used to determine the differences in the number of axons myelinated in the scaffolds, the mean latency and amplitude of cortical motor evoked potentials (CMEPs) and Basso, Beattie & Bresnahan locomotor rating scale (BBB) score. The χ 2 test was used to determine the differences in recovery percentage of CMEPs. Results NSCs survived, but the majority migrated into adjacent host cord and died mostly. Survival rate of NSCs with SCs was higher than that of NSCs without SCs ((1.7831±0.0402)% vs. (1.4911±0.0313)%, P <0.001). Cografted with SCs, NSCs were induced to differentiate towards neurons and might form synaptic connection. The mean number of myelinated axons in PLGA+NSCs+SCs group was more than that in PLGA+NSCs group and in PLGA group ((110.25±30.46) vs. (18.25±3.30) and (11.25±5.54), P <0.01). The percentage of CMEPs recovery in PLGA+NSCs+SCs group was higher than in the other groups (84.8% vs. 50.0% and 37.5%, P <0.05). The amplitude of CMEPs in PLGA+NSCs+SCs group was higher than in the other groups ((1452.63±331.70) μV vs. (428.84±193.01) μV and (117.33±14.40) μV, P <0.05). Ipsilateral retransection resulted in disappearance again and functional loss of CMEPs for a few days. But contralateral retransection completely damaged the bilateral motor function. Conclusions NSCs can survive in PLGA scaffolds, and SCs promote NSCs to survive and differentiate towards neurons in vivo which even might form synaptic connection. The scaffolds seeded with cells facilitate axonal regeneration and myelination and motor function recovery. But regenerating axons have limited contribution to motor function recovery.

Neural Stem Cells in Neurospheres, Embryoid Bodies, and Central Nervous System of Human Embryos

The process of neurogenesis and formation of neural stem cells is reported in human neurospheres (NS) and embryoid bodies (EB) derived from human embryonic stem cells, in vitro, and compared with neural tissue formed in human ectopic embryos in week 4 (stage 9), developed in vivo. This morphological study was done using digital imaging by light microscopy and routine transmission electron microscopy. Both NS and EB form neural rosettes from the surface epithelium much like the process of neural tube formation from ectoderm in the embryo. The rosette is the developmental signature of neuroprogenitors in cultures of differentiating embryonic stem cells and is a radial arrangement of columnar cells that express many of the proteins expressed in neuroepithelial cells in the neural tube. The NS produce all of the major classes of progeny of the neural tube, some of which have been documented here. Specific neural markers expressed in the NS and the clinical implications of this study in cell therapy are also discussed.

19‐Nortestosterone influences neural stem cell proliferation and neurogenesis in the rat brain

Abuse of androgenic anabolic steroids can affect brain function leading to behavioural changes. In this study, the effects of the testosterone analogue, 19-nortestosterone, on rat neural stem cells was examined. The androgen receptor is expressed by cultured embryonic and adult neural stem cells, and is also present in the ventricular epithelium during development and in the adult brain in, among others, dentate gyrus. In neural stem cells stimulated with epidermal growth factor, nandrolone reduced cell proliferation, especially in adult ones. The decrease was abolished by flutamide, a receptor antagonist. Nandrolone also decreased the BrdU labelling of neural stem cells in the dentate gyrus, demonstrating an effect of the hormone on cell proliferation in vivo. The effect of nandrolone was observed with both female and male rats but it was more pronounced in pregnant rats, indicating an involvement of oestrogen in nandrolone action. Nandrolone also decreased the number of newly born neuronal cells in the dentate gyrus of male rats. The results show that nandrolone has important effects on the proliferation and differentiation of neural stem cells expressing the cognate androgen receptor. The data show that the use of nandrolone may severely affect the formation of neural stem cells and could therefore have long-term negative consequences in the brain.

Tramtrack controls glial number and identity in the Drosophila embryonic CNS.

Neurons and glia are often derived from common multipotent stem cells. In Drosophila, neural identity appears to be the default fate of these precursors. Stem cells that generate either neurons or glia transiently express neural stem cell-specific markers. Further development as glia requires the activation of glial-specific regulators. However, this must be accompanied by simultaneous repression of the alternate neural fate. I show that the Drosophila transcriptional repressor Tramtrack is a key repressor of neuronal fates. It is expressed at high levels in all mature glia of the embryonic central nervous system. Analysis of the temporal profile of Tramtrack expression in glia shows that it follows that of existing glial markers. When expressed ectopically before neural stem cell formation, Tramtrack represses the neural stem cell-specific genes asense and deadpan. Surprisingly, Tramtrack protein levels oscillate in a cell cycle-dependent manner in proliferating glia, with expression dropping before replication, but re-initiating after S phase. Overexpression of Tramtrack blocks glial development by inhibiting S-phase and repressing expression of the S-phase cyclin, cyclin E. Conversely, in tramtrack mutant embryos, glia are disrupted and undergo additional rounds of replication. I propose that Tramtrack ensures stable mature glial identity by both repressing neuroblast-specific genes and controlling glial cell proliferation.