Stem Cell Neuronal Differentiation Research Articles

Restoration of hippocampal adult neurogenesis by CDRI-08 (Bacopa monnieri extract) relates with the recovery of BDNF-TrkB levels in male rats with moderate grade hepatic encephalopathy.

Modulation of in vivo adult neurogenesis (AN) is an evolving concept in managing neurodegenerative diseases. CDRI-08, a bacoside-enriched fraction of Bacopa monnieri, has been demonstrated for its neuroprotective actions, but its effect on AN remains unexplored. This article describes the status of AN by monitoring neuronal stem cells (NSCs) proliferation, differentiation/maturation markers and BDNF-TrkB levels (NSCs signalling players) vs. the level of neurodegeneration and their modulations by CDRI-08 in the hippocampal dentate gyrus (DG) of male rats with moderate grade hepatic encephalopathy (MoHE). For NSC proliferation, 10mg/kg b.w. 5-bromo-2'-deoxyuridine (BrdU) was administered i.p. during the last 3days, and for the NSC differentiation study, it was given during the first 3days to the control, the MoHE (developed by 100 mg/kg b.w. of thioacetamide i.p. up to 10days) and to the MoHE male rats co-treated with 350 mg/kg b.w. CDRI-08. Compared with the control rats, the hippocampus DG region of MoHE rats showed significant decreases in the number of Nestin+/BrdU+ and SOX2+/BrdU+ (proliferating) and DCX+/BrdU+ and NeuN+/BrdU+ (differentiating) NSCs. This was consistent with a similar decline in BDNF+/TrkB+ NSCs. However, all these NSC marker positive cells were observed to be recovered to their control levels, with a concordant restoration of total cell numbers in the DG of the CDRI-08-treated MoHE rats. The findings suggest that the restoration of hippocampal AN by CDRI-08 is consistent with the recovery of BDNF-TrkB-expressing NSCs in the MoHE rat model of neurodegeneration.

Viscoelastic cues to induce stem cell migration and neuronal differentiation in cell-free hydrogel-assisted TBI recovery

The concept of tissue-inducing biomaterials, such as osteoinductive biomaterials, has inspired the design of regenerative material with biomimetic cues to manipulate cell/tissue responses but has been little applied for neuroinduction in traumatic brain injury (TBI) treatment. Meanwhile, material design has typically focused on elasticity without viscoelasticity taken into consideration. Here, guided by the intrinsic viscoelasticity of brain tissue and the decisive role of viscoelasticity in cell-matrix interactions, we developed a family of brain-mimicking hydrogels, in which the viscoelasticity can be tuned over a wide range under the premise that hydrogels have a low modulus comparable to that of brain tissue. Then, we revealed the promoted migration of stem cells on the viscoelastic hydrogel resulted from the increased number of motor − clutch pairs and filopodia protrusions, as well as their enhanced neuronal differentiation. In a rat TBI model, the cell-free viscoelastic hydrogel successfully induced endogenous stem cells to migrate into lesions and differentiate into neurons, contributing to brain tissue regeneration and neurological function restoration. This study reveals the great promise of biomimetic viscoelastic matrices for TBI treatment, and simultaneously, provides intriguing insights for the design of tissue-inducing biomaterials.

Salidroside promotes the repair of spinal cord injury by inhibiting astrocyte polarization, promoting neural stem cell proliferation and neuronal differentiation

Spinal cord injury (SCI) remains a formidable challenge, lacking effective treatments. Following SCI, neural stem cells (NSCs) migrate to SCI sites, offering a potential avenue for nerve regeneration, but the effectiveness of this intrinsic repair mechanism remains suboptimal. Salidroside has demonstrated pro-repair attributes in various pathological conditions, including arthritis and cerebral ischemia, and the ability to curtail early-stage inflammation following SCI. However, the specific role of salidroside in the late-stage repair processes of SCI remains less defined. In this investigation, we observed that continuous salidroside treatment in SCI mice improved motor function recovery. Immunofluorescence-staining corroborated salidroside’s capacity to stimulate nerve regeneration and remyelination, suppress glial scar hyperplasia, reduce the activation of neurotoxic A1 astrocytes, and facilitate NSCs migration towards the injured region. Mechanistically, in vitro experiments elucidated salidroside’s significant role in restraining astrocyte proliferation and A1 polarization. It was further established that A1 astrocytes hinder NSCs proliferation while inducing their differentiation into astrocytes. Salidroside effectively ameliorated this inhibition of NSCs proliferation through diminishing c-Jun N-terminal kinase (JNK) pathway phosphorylation and restored their differentiation into neurons by suppressing the signal transducer and activator of transcription 3 (STAT3) pathway. In summary, our findings suggest that salidroside holds promise as a therapeutic agent for traumatic SCI treatment.

Alternative splicing of a chromatin modifier alters the transcriptional regulatory programs of stem cell maintenance and neuronal differentiation

Development of embryonic stem cells (ESCs) into neurons requires intricate regulation of transcription, splicing, and translation, but how these processes interconnect is not understood. We found that polypyrimidine tract binding protein 1 (PTBP1) controls splicing of DPF2, a subunit of BRG1/BRM-associated factor (BAF) chromatin remodeling complexes. Dpf2 exon 7 splicing is inhibited by PTBP1 to produce the DPF2-S isoform early in development. During neuronal differentiation, loss of PTBP1 allows exon 7 inclusion and DPF2-L expression. Different cellular phenotypes and gene expression programs were induced by these alternative DPF2 isoforms. We identified chromatin binding sites enriched for each DPF2 isoform, as well as sites bound by both. In ESC, DPF2-S preferential sites were bound by pluripotency factors. In neuronal progenitors, DPF2-S sites were bound by nuclear factor I (NFI), while DPF2-L sites were bound by CCCTC-binding factor (CTCF). DPF2-S sites exhibited enhancer modifications, while DPF2-L sites showed promoter modifications. Thus, alternative splicing redirects BAF complex targeting to impact chromatin organization during neuronal development.

Mild hypothermia promotes neuronal differentiation of human neural stem cells via RBM3-SOX11 signaling pathway

Mild hypothermia promotes neuronal differentiation of human neural stem cells via RBM3-SOX11 signaling pathway

Lipotype acquisition during neural development is not recapitulated in stem cell-derived neurons.

During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery during development, but when and how this occurs is unclear. To address this, we used high-resolution MSALL lipidomics to generate an extensive time-resolved resource of mouse brain development covering early embryonic and postnatal stages. This revealed a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. Using the resource as a reference, we next examined to which extent this can be recapitulated by commonly used protocols for in vitro neuronal differentiation of stem cells. Here, we found that the programming of the lipid metabolic machinery is incomplete and that stem cell-derived cells can only partially acquire a neural lipotype when the cell culture media is supplemented with brain-specific lipid precursors. Altogether, our work provides an extensive lipidomic resource for early mouse brain development and highlights a potential caveat when using stem cell-derived neuronal progenitors for mechanistic studies of lipid biochemistry, membrane biology and biophysics, which nonetheless can be mitigated by further optimizing in vitro differentiation protocols.

Protein S-nitrosylation is involved in valproic acid-promoted neuronal differentiation of adipose tissue-derived stem cells

Neuronal differentiation of adipose tissue-derived stem cells (ASCs) is greatly promoted by valproic acid (VPA) with cAMP elevating agents thorough NO signaling pathways, but its mechanism is not fully understood. In the present study, we investigate the involvement of protein S-nitrosylation in the VPA-promoted neuronal differentiation of ASCs. The whole amount of S-nitrosylated protein was increased by the treatment with VPA alone for three days in ASCs. An inhibitor of thioredoxin reductase (TrxR), auranofin, further increased the amount of S-nitrosylated protein and enhances the VPA-promoted neuronal differentiation in ASCs. On the contrary, another inhibitor of TrxR, dinitrochlorobenzene, inhibited the VPA-promoted neuronal differentiation in ASCs even with cAMP elevating agents, which was accompanied by unexpectedly decreased S-nitrosylated protein. It was considered from these results that increased protein S-nitrosylation is involved in VPA-promoted neuronal differentiation of ASCs. By the proteomic analysis of S-nitrosylated protein in VPA-treated ASCs, no identified proteins could be specifically related to VPA-promoted neuronal differentiation. The identified proteins, however, included those involved in the metabolism of substances regulating neuronal differentiation, such as aspartate and glutamate.

Aquaporin-4 and transient receptor potential vanilloid 4 balance in early postnatal neurodevelopment.

In the adult brain, the water channel aquaporin-4 (AQP4) is expressed in astrocyte endfoot, in supramolecular assemblies, called "Orthogonal Arrays of Particles" (OAPs) together with the transient receptor potential vanilloid 4 (TRPV4), finely regulating the cell volume. The present study aimed at investigating the contribution of AQP4 and TRPV4 to CNS early postnatal development using WT and AQP4 KO brain and retina and neuronal stem cells (NSCs), as an in vitro model of astrocyte differentiation. Western blot analysis showed that, differently from AQP4 and the glial cell markers, TRPV4 was downregulated during CNS development and NSC differentiation. Blue native/SDS-PAGE revealed that AQP4 progressively organized into OAPs throughout the entire differentiation process. Fluorescence quenching assay indicated that the speed of cell volume changes was time-related to NSC differentiation and functional to their migratory ability. Calcium imaging showed that the amplitude of TRPV4 Ca2+ transient is lower, and the dynamics are changed during differentiation and suppressed in AQP4 KO NSCs. Overall, these findings suggest that early postnatal neurodevelopment is subjected to temporally modulated water and Ca2+ dynamics likely to be those sustaining the biochemical and physiological mechanisms responsible for astrocyte differentiation during brain and retinal development.

Comparative evaluation of divergent concoction of NGF, BDNF, EGF, and FGF growth factor's role in enhancing neuronal differentiation of adipose-derived mesenchymal stem cells

MSCs (Mesenchymal Stem Cells) can differentiate into various lineages, including neurons and glial cells. In the past few decades, MSCs have been well explored in the context of neuronal differentiation and have been reported to have the immense potential to form distinct kinds of neurons. The distinguishing features of MSCs make them among the most desired cell sources for stem cell therapy. This study involved the trans-differentiation of Adipose-derived human Mesenchymal Stem Cells (ADMSCs) into neurons. The protocol employs a cocktail of chemical inducers in different combinations, including Brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), and Nerve growth factor (NGF) Fibroblastic growth factor (FGF), in induction media. Both types have been successfully differentiated into neurons, confirmed by morphological aspects and the presence of neural-specific markers through RT-PCR (Reverse transcription polymerase chain reaction) studies and immunocytochemistry assay. They have shown excellent morphology with long neurites, synaptic connections, and essential neural markers to validate their identity. The results may significantly contribute to cell replacement therapy for neurological disorders.

Aβ oligomers reduce neuronal plasticity in an aged animal model of Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) is characterized by the extracellular accumulation of senile plaques composed of beta‐amyloid (Aβ). Aβ oligomers (AβO) induce damage beginning with loss of synapses, followed by neurite network disorganization and neuronal death. The hippocampus is a cerebral structure and a major place of neuroplasticity and a key structure for memory processing and consolidation. Hippocampal atrophy is well documented in AD. Neuronal plasticity persists during ageing and could adaptative response to cognitive decline during ageing or dementia.Alterations in adult neuronal plasticity and synaptic dysfunctions are detected in rodent models of Alzheimer’s disease. Thus, early dysfunction in neural stem cell production and neuronal differentiation could be linked to reduced neuronal plasticity in AD.MethodIn this study, to investigate the effect of age on neurogenesis, in young (12 weeks), aged and healthy (16 months) mice and in an in vivo model of AD, based on intra‐hippocampal injection of a AβO/protofibril preparation in aged (16 months) mice.Proliferation of cells (BrdU, Ki67), differentiation (DCX) and maturation (NeuN) into neurons were studied in the dendate gyrus of mice. Activation of glial cells was also assessed by immunohistochemistry. In addition, cognitive performance of mice were assessed using the Y‐maze test (short‐term spatial memory) in the same animal.ResultOur results show a decrease in neurogenesis in aged mice compared to young mice. Injection of AβO in the CA1 area was also associated with decreased neurogenesis in the dentate gyrus, neuroinflammation and to reduce performance in the Y‐maze test.This reduction in neuroblast generation was revealed by a decrease in the number of BrdU(+), Ki67(+) and DCX(+) cells in the dentate gyrus.ConclusionWe evidenced a decreased in neuronal plasticity in aging which is exacerbated in our in vivo model of AD and connected to cognitive decline.

Expression of Major Histocompatibility Complex during Neuronal Differentiation of Somatic Cell Nuclear Transfer-Human Embryonic Stem Cells.

Human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs), induced pluripotent stem cells, and somatic cell nuclear transfer (SCNT)-hESCs can permanently self-renew while maintaining their capacity to differentiate into any type of somatic cells, thereby serving as an important cell source for cell therapy. However, there are persistent challenges in the application of hPSCs in clinical trials, where one of the most significant is graft rejection by the patient immune system in response to human leukocyte antigen (HLA) mismatch when transplants are obtained from an allogeneic (non-self) cell source. Homozygous SCNT-hESCs (homo-SCNT-hESCs) were used to simplify the clinical application and to reduce HLA mismatch. Here, we present a xeno-free protocol that confirms the efficient generation of neural precursor cells in hPSCs and also the differentiation of dopaminergic neurons. Additionally, there was no difference when comparing the HLA expression patterns of hESC, homo-SCNT-hESCs and hetero-SCNT-hESCs. We propose that there are no differences in the differentiation capacity and HLA expression among hPSCs that can be cultured in vitro. Thus, it is expected that homo-SCNT-hESCs will possess a wider range of applications when transplanted with neural precursor cells in the context of clinical trials.

Leptin Promotes the Proliferation and Neuronal Differentiation of Neural Stem Cells through the Cooperative Action of MAPK/ERK1/2, JAK2/STAT3 and PI3K/AKT Signaling Pathways.

The potential of neural stem cells (NSCs) for neurological disorders the treatment has relied in large part upon identifying the NSCs fate decision. The hormone leptin has been reported to be a crucial regulator of brain development, able to influence the glial and neural development, yet, the underlying mechanism of leptin acting on NSCs' biological characteristics is still poorly understood. This study aims to investigate the role of leptin in the biological properties of NSCs. In this study, we investigate the possibility that leptin may regulate the NSCs' fate decision, which may promote the proliferation and neuronal differentiation of NSCs and thus act positively in neurological disorders. NSCs from the embryonic cerebral cortex were used in this study. We used CCK-8 assay, ki67 immunostaining, and FACS analysis to confirm that 25-100 ng/mL leptin promotes the proliferation of NSCs in a concentration-dependent pattern. This change was accompanied by the upregulation of p-AKT and p-ERK1/2, which are the classical downstream signaling pathways of leptin receptors b (LepRb). Inhibition of PI3K/AKT or MAPK/ERK signaling pathways both abolished the effect of leptin-induced proliferation. Moreover, leptin also enhanced the directed neuronal differentiation of NSCs. A blockade of the PI3K/AKT pathway reversed leptin-stimulated neurogenesis, while a blockade of JAK2/STAT3 had no effect on it. Taken together, our results support a role for leptin in regulating the fate of NSCs differentiation and promoting NSCs proliferation, which could be a promising approach for brain repair via regulating the biological characteristics of NSCs.

Function of GSK‑3 signaling in spinal cord injury (Review).

Spinal cord injury (SCI) is a major social problem with a heavy burden on patient physiology and psychology. Glial scar formation and irreversible neuron loss are the two key points during SCI progression. During the acute phase of spinal cord injury, glial scars form, limiting the progression of inflammation. However, in the subacute or chronic phase, glial scarring inhibits axon regeneration. Following spinal cord injury, irreversible loss of neurons leads to further aggravation of spinal cord injury. Several therapies have been developed to improve either glial scar or neuron loss; however, few therapies reach the stage of clinical trials and there are no mainstream therapies for SCI. Exploring the key mechanism of SCI is crucial for finding further treatments. Glycogen synthase kinase-3 (GSK-3) is a widely expressed kinase with important physiological and pathophysiological functions in vivo. Dysfunction of the GSK-3 signaling pathway during SCI has been widely discussed for controlling neurite growth in vitro and in vivo, improving the proliferation and neuronal differentiation of endogenous neural stem cells and functional recovery from spinal cord injury. SCI can decrease the phosphorylated (p)/total (t)-GSK-3β ratio, which leads to an increase in apoptosis, whereas treatment with GSK-3 inhibitors can promote neurogenesis. In addition, several therapies for the treatment of SCI involve signaling pathways associated with GSK-3. Furthermore, signaling pathways associated with GSK-3 also participate in the pathological process of neuropathic pain that remains following SCI. The present review summarized the roles of GSK-3 signaling in SCI to aid in the understanding of GSK-3 signaling during the pathological processes of SCI and to provide evidence for the development of comprehensive treatments.

Mettl1-mediated internal m7G methylation of Sptbn2 mRNA elicits neurogenesis and anti-alzheimer’s disease

BackgroundN7-methylguanosine (m7G) is one of the most conserved modifications in nucleosides impacting mRNA export, splicing, and translation. However, the precise function and molecular mechanism of internal mRNA m7G methylation in adult hippocampal neurogenesis and neurogenesis-related Alzheimer’s disease (AD) remain unknown.ResultsWe profiled the dynamic Mettl1/Wdr4 expressions and m7G modification during neuronal differentiation of neural stem cells (NSCs) in vitro and in vivo. Adult hippocampal neurogenesis and its molecular mechanisms were examined by morphology, biochemical methods and biological sequencing. The translation efficiency of mRNA was detected by polysome profiling. The stability of Sptbn2 mRNA was constructed by RNA stability assay. APPswe/PS1ΔE9 (APP/PS1) double transgenic mice were used as model of AD. Morris water maze was used to detect the cognitive function.MethodsWe found that m7G methyltransferase complex Mettl1/Wdr4 as well as m7G was significantly elevated in neurons. Functionally, silencing Mettl1 in neural stem cells (NSCs) markedly decreased m7G modification, neuronal genesis and proliferation in addition to increasing gliogenesis, while forced expression of Mettl1 facilitated neuronal differentiation and proliferation. Mechanistically, the m7G modification of Sptbn2 mRNA by Mettl1 enhanced its stability and translation, which promoted neurogenesis. Importantly, genetic defciency of Mettl1 reduced hippocampal neurogenesis and spatial memory in the adult mice. Furthermore, Mettl1 overexpression in the hippocampus of APP/PS1 mice rescued neurogenesis and behavioral defects.ConclusionOur findings unravel the pivotal role of internal mRNA m7G modification in Sptbn2-mediated neurogenesis, and highlight Mettl3 regulation of neurogenesis as a novel therapeutic target in AD treatment.

Direct deep UV lithography to micropattern PMMA for stem cell culture.

Microengineering is increasingly being used for controlling the microenvironment of stem cells. Here, a novel method for fabricating structures with subcellular dimensions in commonly available thermoplastic poly(methyl methacrylate) (PMMA) is shown. Microstructures are produced in PMMA substrates using Deep Ultraviolet lithography, and the effect of different developers is described. Microgrooves fabricated in PMMA are used for the neuronal differentiation of mouse embryonic stem cells (mESCs) directly on the polymer. The fabrication of 3D, curvilinear patterned surfaces is also highlighted. A 3D multilayered microfluidic chip is fabricated using this method, which includes a porous polycarbonate (PC) membrane as cell culture substrate. Besides directly manufacturing PMMA-based microfluidic devices, an application of the novel approach is shown where a reusable PMMA master is created for replicating microstructures with polydimethylsiloxane (PDMS). As an application example, microchannels fabricated in PDMS are used to selectively expose mESCs to soluble factors in a localized manner. The described microfabrication process offers a remarkably simple method to fabricate for example multifunctional topographical or microfluidic culture substrates outside cleanrooms, thereby using inexpensive and widely accessible equipment. The versatility of the underlying process could find various applications also in optical systems and surface modification of biomedical implants.

CHI3L1 signaling impairs hippocampal neurogenesis and cognitive function in autoimmune-mediated neuroinflammation.

Chitinase-3-like protein 1 (CHI3L1) is primarily secreted by activated astrocytes in the brain and is known as a reliable biomarker for inflammatory central nervous system (CNS) conditions such as neurodegeneration and autoimmune disorders like neuromyelitis optica (NMO). NMO is an astrocyte disease caused by autoantibodies targeting the astroglial protein aquaporin 4 (AQP4) and leads to vision loss, motor deficits, and cognitive decline. In this study examining CHI3L1's biological function in neuroinflammation, we found that CHI3L1 expression correlates with cognitive impairment in our NMO patient cohort. Activated astrocytes secrete CHI3L1 in response to AQP4 autoantibodies, and this inhibits the proliferation and neuronal differentiation of neural stem cells. Mouse models showed decreased hippocampal neurogenesis and impaired learning behaviors, which could be rescued by depleting CHI3L1 in astrocytes. The molecular mechanism involves CHI3L1 engaging the CRTH2 receptor and dampening β-catenin signaling for neurogenesis. Blocking this CHI3L1/CRTH2/β-catenin cascade restores neurogenesis and improves cognitive deficits, suggesting the potential for therapeutic development in neuroinflammatory disorders.

KLF2 Regulates Neural Differentiation of Dental Pulp-derived Stem Cells by Modulating Autophagy and Mitophagy.

Transplantation of stem cells for treating neurodegenerative disorders is a promising future therapeutic approach. However, the molecular mechanism underlying the neuronal differentiation of dental pulp-derived stem cells (DPSC) remains inadequately explored. The current study aims to define the regulatory role of KLF2 (Kruppel-like factor 2) during the neural differentiation (ND) of DPSC. We first investigated the transcriptional and translational expression of KLF2, autophagy, and mitophagy-associated markers during the ND of DPSC by using quantitative RT-PCR and western blot methods. After that, we applied the chemical-mediated loss- and gain-of-function approaches using KLF2 inhibitor, GGPP (geranylgeranyl pyrophosphate), and KLF2 activator, GGTI-298 (geranylgeranyl transferase inhibitor-298) to delineate the role of KLF2 during ND of DPSC. The western blot, qRT-PCR, and immunocytochemistry were performed to determine the molecular changes during ND after KLF2 deficiency and KLF2 sufficiency. We also analyzed the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer. Our study demonstrated that the expression level of KLF2, autophagy, and mitophagy-associated markers were significantly elevated during the ND of DPSC. Next, we found that the KLF2 inhibitor, GGPP significantly reduced the ND of DPSC. Inversely, KLF2 overexpression accelerated the molecular phenomenon of DPSC's commitment towards ND, indicating the crucial role of KLF2 in neurogenesis. Moreover, we found that the KLF2 positively regulated autophagy, mitophagy, and the Wnt5a signaling pathway during neurogenesis. Seahorse XFe24 analysis revealed that the ECAR and OCR parameters were significantly increased during ND, and inhibition of KLF2 marginally reversed them towards DPSC's cellular bioenergetics. However, KLF2 overexpression shifted the cellular energy metabolism toward the quiescent stage. Collectively, our findings provide the first evidence that the KLF2 critically regulates the neurogenesis of DPSC by inducing autophagy and mitophagy.

M2 Microglia Extracellular Vesicle miR-124 Regulates Neural Stem Cell Differentiation in Ischemic Stroke via AAK1/NOTCH.

Small extracellular vesicles (sEVs) derived from M2 microglia (M2-microglia-derived small extracellular vesicles [M2-sEVs]) contribute to central nervous system repair, although the underlying mechanism remains unknown. In this study, we aimed to identify the mechanism through which microRNA-124 (miR-124) carried in sEVs promotes neural stem cell (NSC) proliferation and neuronal differentiation in the ischemic mouse brain. M2-sEVs with or without miR-124 knockdown were injected intravenously for 7 consecutive days after transient middle cerebral artery occlusion surgery. The atrophy volume, neurological score, and degree of neurogenesis were examined at different time points after ischemic attack. NSCs treated with different sEVs were subjected to proteomic analysis. Target protein concentrations were quantified, and subsequent bioinformatic analysis was conducted to explore the key signaling pathways. M2-sEV transplantation promoted functional neurological recovery following transient middle cerebral artery occlusion injury. M2-sEV treatment decreased the brain atrophy volume, neurological score, and mortality rate. The effect was reserved by knockdown of miR-124 in M2-sEVs. M2-sEVs promoted proliferation and differentiation of mature neuronal NSCs in vivo. Proteomic analysis of NSC samples treated with M2-sEVs with and without miR-124 knockdown revealed that AAK1 (adaptor-associated protein kinase 1) was the key responding protein in NSCs. The binding of AAK1 to Notch promoted the differentiation of NSCs into neurons rather than astrocytes. Our data suggest that AAK1/Notch is the key pathway in NSCs that responds to the miR-124 carried within M2-sEVs in the ischemic brain. M2-sEVs carrying ample quantities of miR-124 promote functional recovery after ischemic stroke by enhancing NSC proliferation and differentiation. Targeting of M2-sEVs could represent a potential therapeutic strategy for brain recovery.

MiR-199a-5p enhances neuronal differentiation of neural stem cells and promotes neurogenesis by targeting Cav-1 after cerebral ischemia.

MicroRNAs (miRs) are involved in endogenous neurogenesis, enhancing of which has been regarded as a potential therapeutic strategy for ischemic stroke treatment; however, whether miR-199a-5p mediates postischemic neurogenesis remains unclear. This study aims to investigate the proneurogenesis effects of miR-199a-5p and its possible mechanism after ischemic stroke. Neural stem cells (NSCs) were transfected using Lipofectamine 3000 reagent, and the differentiation of NSCs was evaluated by immunofluorescence and Western blotting. Dual-luciferase reporter assay was performed to verify the target gene of miR-199a-5p. MiR-199a-5p agomir/antagomir were injected intracerebroventricularly. The sensorimotor functions were evaluated by neurobehavioral tests, infarct volume was measured by toluidine blue staining, neurogenesis was detected by immunofluorescence assay, and the protein levels of neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), caveolin-1 (Cav-1), vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF) were measured by Western blotting. MiR-199a-5p mimic enhanced neuronal differentiation and inhibited astrocyte differentiation of NSCs, while a miR-199a-5p inhibitor induced the opposite effects, which can be reversed by Cav-1 siRNA. Cav-1 was through the dual-luciferase reporter assay confirmed as a target gene of miR-199a-5p. miR-199a-5p agomir in rat stroke models manifested multiple benefits, such as improving neurological deficits, reducing infarct volume, promoting neurogenesis, inhibiting Cav-1, and increasing VEGF and BDNF, which was reversed by the miR-199a-5p antagomir. MiR-199a-5p may target and inhibit Cav-1 to enhance neurogenesis and thus promote functional recovery after cerebral ischemia. These findings indicate that miR-199a-5p is a promising target for the treatment of ischemic stroke.

Inorganic nanorods direct neuronal differentiation of bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent cells and are considered a potential source for tissue and organ repair due to their self-renewal, proliferation, and differentiation abilities. However, in most cases, MSCs are needed to be stimulated with external growth factors to promote their proliferation and differentiation. Over the past decade, it has been demonstrated that nanomaterials could facilitate MSC proliferation and differentiation, and excellent efforts are carried out to investigate their possible modulating pattern and mechanisms for MSC differentiation. Europium hydroxide (EuIII(OH)3) nanorods (EHN) are well-researched for their biomimicking properties and act as a substitute for growth factors that induce cell proliferation, migration, and differentiation. In the current study, the human MSCs were chosen as an in vitro model for evaluating the role of EHN in modulating the differentiation process of MSCs into neuronal and glial lineages. The characterization of MSCs and differentiated neuronal cells observed by flow cytometry, confocal, and gene marker expression studies supported our hypothesis that the EHNs are pro-angiogenic and pro-neurogenic. Finally, altogether our results suggest that EHNs have the potential to play an essential part in developing novel treatment strategies for neurodegenerative diseases and spinal cord injuries based on the nanomedicine approach.