Retinoic acid induced specific changes in the phosphoproteome of C17.2 neural stem cells.

Retinoic acid (RA), a vitamin A derivative, is an effective cell differentiating factor which plays critical roles in neuronal differentiation induction and the production of neurotransmitters in neurons. However, the specific changes in phosphorylation levels and downstream signalling pathways associated with RA remain unclear. This study employed qualitative and quantitative phosphoproteomics approaches based on mass spectrometry to investigate the phosphorylation changes induced by RA in C17.2 neural stem cells (NSCs). Dimethyl labelling, in conjunction with TiO2 phosphopeptide enrichment, was utilized to profile the phosphoproteome of self-renewing and RA-induced differentiated cells in C17.2 NSCs. The results of our study revealed that, qualitatively, 230 and 14 phosphoproteins were exclusively identified in the self-renewal and RA-induced groups respectively. Quantitatively, we successfully identified and quantified 177 unique phosphoproteins, among which 70 exhibited differential phosphorylation levels. Analysis of conserved phosphorylation motifs demonstrated enrichment of motifs corresponding to cyclin-dependent kinase and MAPK in the RA-induced group. Additionally, through a comprehensive literature and database survey, we found that the differentially expressed proteins were associated with the Wnt/ß-catenin and Hippo signalling pathways. This work sheds light on the changes in phosphorylation levels induced by RA in C17.2 NSCs, thereby expanding our understanding of the molecular mechanisms underlying RA-induced neuronal differentiation.

Comprehensive in-silico analysis of deleterious SNPs in APOC2 and APOA5 and their differential expression in cancer and cardiovascular diseases conditions.

Genetic variations in APOC2 and APOA5 genes involve activating lipoprotein lipase (LPL), responsible for the hydrolysis of triglycerides (TG) in blood and whose impaired functions affect the TG metabolism and are associated with metabolic diseases. In this study, we investigate the biological significance of genetic variations at the DNA sequence and structural level using various computational tools. Subsequently, 8 (APOC2) and 17 (APOA5) non-synonymous SNPs (nsSNPs) were identified as high-confidence deleterious SNPs based on the effects of the mutations on protein conservation, stability, and solvent accessibility. Furthermore, based on our docking results, the interaction of native and mutant forms of the corresponding proteins with LPL depicts differences in root mean square deviation (RMSD), and binding affinities suggest that these mutations may affect their function. Furthermore, in vivo, and in vitro studies have shown that differential expression of these genes in disease conditions due to the influence of nsSNPs abundance may be associated with promoting the development of cancer and cardiovascular diseases. Preliminary screening using computational methods can be a helpful start in understanding the effects of mutations in APOC2 and APOA5 on lipid metabolism; however, further wet-lab experiments would further strengthen the conclusions drawn from the computational study.

A phosphoproteomics study reveals a defined genetic program for neural lineage commitment of neural stem cells induced by olfactory ensheathing cell-conditioned medium.

Since both Olfactory ensheathing cells (OECs) and neural stem cells (NSCs) have shown certain efficacy in the cellular therapy of nerve injury and disease, there have been a series of investigations in recent years looking at the co-culture of NSCs and OECs. Protein phosphorylation forms the basis for identifying a variety of cellular signaling pathways responsible for regulating the self-renewal and differentiation of NSCs induced by OECs. To better understand the signaling cascades in the early phases of OEC-induced NSC differentiation, changes in the NSC proteome and phosphoproteome during the first 24 h were determined using dimethyl labeling and TiO2 phosphorylation enrichment coupled with Liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 565 proteins and 2511 phosphorylation sites were identified. According to quantitative phosphoproteomics analyses of NSC differentiation induced by OECs during the first 12 and 24 h, it was speculated that there were at least two different signal waves: one peaking within 12 h after stimulation and the second upsurge after 24 h. In addition to understanding the dynamics of the proteome and phosphoproteome in the early stages of NSC differentiation, our analyses identified a key role of the TGF-ß3 protein secreted by OECs, which may be an initiating factor that promotes differentiation of NSCs into neurons induced by OECs. These findings not only redemonstrated a OECs-based therapeutic strategy in cell therapy, but also added a node to the regulatory network for the neural lineage commitment of NSCs induced by OECs.

Extracellular vesicles derived from hypoxia-preconditioned olfactory mucosa mesenchymal stem cells enhance angiogenesis via miR-612.

Mesenchymal stem cells (MSCs) play important roles in tissue repair and regeneration, such as the induction of angiogenesis, particularly under hypoxic conditions. However, the molecular mechanisms underlying hypoxic MSC activation remain largely unknown. MSC-derived extracellular vesicles (EVs) are vital mediators of cell-to-cell communication and can be directly utilized as therapeutic agents for tissue repair and regeneration. Here, we explored the effects of EVs from human hypoxic olfactory mucosa MSCs (OM-MSCs) on angiogenesis and its underlying mechanism. EVs were isolated from normoxic (N) OM-MSCs (N-EVs) and hypoxic (H) OM-MSCs (H-EVs) using differential centrifugation and identified by transmission electron microscopy and flow cytometry. In vitro and in vivo, both types of OM-MSC-EVs promoted the proliferation, migration, and angiogenic activities of human brain microvascular endothelial cells (HBMECs). In addition, angiogenesis-stimulatory activity in the H-EV group was significantly enhanced compared to the N-EV group. MicroRNA profiling revealed a higher abundance of miR-612 in H-EVs than in N-EVs, while miR-612 inactivation abolished the N-EV treatment benefit. To explore the roles of miR-612, overexpression and knock-down experiments were performed using a mimic and inhibitor or agomir and antagomir of miR-612. The miR-612 target genes were confirmed using the luciferase reporter assay. Gain- and loss-of-function studies allowed the validation of miR-612 (enriched in hypoxic OM-MSC-EVs) as a functional messenger that stimulates angiogenesis and represses the expression of TP53 by targeting its 3'-untranslated region. Further functional assays showed that hypoxic OM-MSC-EVs promote paracrine Hypoxia-inducible factor 1-alpha (HIF-1α)-Vascular endothelial growth factor (VEGF) signaling in HBMECs via the exosomal miR-612-TP53-HIF-1α-VEGF axis. These findings suggest that hypoxic OM-MSC-EVs may represent a promising strategy for ischemic disease by promoting angiogenesis via miR-612 transfer.

[Hypoxic condition promotes olfactory mucosa mesenchymal stem cells to differentiate into neurons and underlying mechanisms].

OBJECTIVE: To explore whether hypoxic condition could promote the olfactory mucosa mesenchymal stem cells (OM-MSCs) to differentiate into neurons with the olfactory ensheathing cells (OECs) supernatant and the potential mechanisms.
 Methods: The OM-MSCs and OECs were isolated and cultured, and they were identified by flow cytometry and immunofluorescence. The OM-MSCs were divided into three groups: a 3%O2+ HIF-1α inhibitors (lificiguat: YC-1) + OECs supernatant group (Group A) , a 3%O2 + OECs supernatant group (Group B) and a 21%O2 + OECs supernatant group (Control group). The neurons, which were differentiated from OM-MSCs, were assessed by immunofluorescence test. The mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α), ßIII-tubulin and glial fibrillary acidic portein (GFAP) were detected by quantitative polymerase chain reaction (Q-PCR) and Western blot. The potassium channels were analyzed by patch clamp.
 Results: The neurons differentiated from OM-MSCs expressed the most amount of ßIII-tubulin, and the result of Q-PCR showed that HIF-1α expression in the Group B was significantly higher than that in the other groups (all P<0.05). Western blot result showed that the ßIII-tubulin protein expression was significantly higher and GFAP protein expression was obviously decreased in the Group B (both P<0.05). The patch clamp test confirmed that the potassium channels in the neurons were activated.
 Conclusion: Hypoxic condition can significantly increase the neuronal differentiation of OM-MSCs by the OECs supernatant and decrease the production of neuroglia cells, which is associated with the activation of HIF-1 signal pathway.

[Biological characteristics of human olfactory mucosa mesenchymal stem cells].

OBJECTIVE: To observe the biological characteristics of the human olfactory mucosa mesenchymal stem cells (hOM-MSCs). METHODS: The hOM-MSCs were isolated, cultured and identified in vitro. Scanning electron microscope and transmission electron microscope were used to observe the ultrastructure of hOMMSCs. Th e cells were induced towards adipocyte, osteocyte, neural stem cells, neural-like-cells in vitro. RESULTS: The hOM-MSCs were mainly in spindle shape, arranged with radial colony. The hOMMSCs expressed CD73 and CD90 but no CD34 and CD45. Th e short and thick microvilli processes were seen at the surface of hOM-MSCs by scanning electron microscope, and 2 different cellular morphology of hOM-MSCs were seen under transmission electron microscope. Moreover, the hOMMSCs could be differentiated into adipocyte, osteocyte, neural stem cells and neural cells. CONCLUSION: The hOM-MSCs possess general biological characteristics of MSCs and display multiple differentiation functions. They can be served as ideal seed cells in tissue-engineering for injury repair.

Effects of bone marrow mesenchymal stromal cells-derived therapies for experimental traumatic brain injury： A meta-analysis.

Background: Bone-marrow-derived mesenchymal stromal (stem) cells [also called MSC(M)] and their extracellular vesicles (EVs) are considered a potentially innovative form of therapy for traumatic brain injury (TBI). Nevertheless, their application to TBI particularly remains preclinical, and the effects of these cells remain unclear and controversial. Therefore, an updated meta-analysis of preclinical studies is necessary to assess the effectiveness of MSC(M) and MSC(M) derived EVs in clinical trials. Methods: The following databases were searched (to December 2022): PubMed, Web of Science, and Embase. In this study, we measured functional outcomes based on the modified neurological severity score (mNSS), cognitive outcomes based on the Morris water maze (MWM), and histopathological outcomes based on lesion volume. A random effects meta-analysis was conducted to evaluate the effect of mNSS, MWM, and lesion volume. Results: A total of 2163 unique records were identified from our search, with Fifty-five full-text articles satisfying inclusion criteria. A mean score of 5.75 was assigned to the studies' quality scores, ranging from 4 to 7. MSC(M) and MSC(M) derived EVs had an overall positive effect on the mNSS score and MWM with SMDs -2.57 (95 % CI -3.26; -1.88; p < 0.01) and - 2.98 (95 % CI -4.21; -1.70; p < 0.01), respectively. As well, MSC(M) derived EVs were effective in reducing lesion volume by an SMD of - 0.80 (95 % CI -1.20; -0.40; p < 0.01). It was observed that there was significant variation among the studies, but further analyses could not determine the cause of this heterogeneity. Conclusions: MSC(M) and MSC(M) derived EVs are promising treatments for TBI in pre-clinical studies, and translation to the clinical domain appears warranted. Besides, large-scale trials in animals and humans are required to support further research due to the limited sample size of MSC(M) derived EVs.

TAK1 inhibition mitigates intracerebral hemorrhage-induced brain injury through reduction of oxidative stress and neuronal pyroptosis via the NRF2 signaling pathway.

Introduction: Intracerebral hemorrhage (ICH) often triggers oxidative stress through reactive oxygen species (ROS). Transforming growth factor-ß-activated kinase 1 (TAK1) plays a pivotal role in regulating oxidative stress and inflammation across various diseases. 5Z-7-Oxozeaenol (OZ), a specific inhibitor of TAK1, has exhibited therapeutic effects in various conditions. However, the impact of OZ following ICH and its underlying molecular mechanisms remain elusive. This study aimed to explore the possible role of OZ in ICH and its underlying mechanisms by inhibiting oxidative stress-mediated pyroptosis. Methods: Adult male Sprague-Dawley rats were subjected to an ICH model, followed by treatment with OZ. Neurobehavioral function, blood-brain barrier integrity, neuronal pyroptosis, and oxidative stress markers were assessed using various techniques including behavioral tests, immunofluorescence staining, western blotting, transmission electron microscopy, and biochemical assays. Results: Our study revealed that OZ administration significantly inhibited phosphorylated TAK1 expression post-ICH. Furthermore, TAK1 blockade by OZ attenuated blood-brain barrier (BBB) disruption, neuroinflammation, and oxidative damage while enhancing neurobehavioral function. Mechanistically, OZ administration markedly reduced ROS production and oxidative stress by facilitating nuclear factor-erythroid 2-related factor 2 (NRF2) nuclear translocation. This was accompanied by a subsequent suppression of the NOD-like receptor protein 3 (NLRP3) activation-mediated inflammatory cascade and neuronal pyroptosis. Discussion: Our findings highlight that OZ alleviates brain injury and oxidative stress-mediated pyroptosis via the NRF2 pathway. Inhibition of TAK1 emerges as a promising approach for managing ICH.

Mesenchymal Stem Cell Senescence during Aging:From Mechanisms to Rejuvenation Strategies.

In cell transplantation therapy, mesenchymal stem cells(MSCs)are ideal seed cells due to their easy acquisition and cultivation, strong regenerative capacity, multi-directional differentiation abilities, and immunomodulatory effects. Autologous MSCs are better applicable compared with allogeneic MSCs in clinical practice. The elderly are the main population for cell transplantation therapy, but as donor aging, MSCs in the tissue show aging-related changes. When the number of generations of in vitro expansion is increased, MSCs will also exhibit replicative senescence. The quantity and quality of MSCs decline during aging, which limits the efficacy of autologous MSCs transplantation therapy. In this review, we examine the changes in MSC senescence as a result of aging, discuss the progress of research on mechanisms and signalling pathways of MSC senescence, and discuss possible rejuvenation strategies of aged MSCs to combat senescence and enhance the health and therapeutic potential of MSCs.

Therapeutic potential of stem cell extracellular vesicles for ischemic stroke in preclinical rodent models: a meta-analysis.

BACKGROUND: Extracellular vesicles derived from stem cells (SC-EVs) have been proposed as a novel therapy for ischemic stroke. However, their effects remain incompletely understood. Therefore, we conducted this meta-analysis to systematically review the efficacy of SC-EVs on ischemic stroke in preclinical rodent models. METHODS: Using PubMed, EMBASE, and the Web of Science, we searched through studies published up to August 2021 that investigated the treatment effects of SC-EVs in a rodent ischemic stroke model. Infarct volume was the primary outcome. Neurological severity scores (mNSS) were the secondary outcome. The standard mean difference (SMD) and the confidence interval (CI) were calculated using a random-effects model. R and Stata 15.1 were used to conduct the meta-analysis. RESULTS: Twenty-one studies published from 2015 to 2021 met the inclusion criteria. We also found that SCs-EVs reduced infarct volume by an SMD of - 2.05 (95% CI - 2.70, - 1.40; P < 0.001). Meanwhile, our results revealed an overall positive effect of SCs-derived EVs on the mNSS with an SMD of - 1.42 (95% CI - 1.75, - 1.08; P < 0.001). Significant heterogeneity among studies was observed. Further stratified and sensitivity analyses did not identify the source of heterogeneity. CONCLUSION: The present meta-analysis confirmed that SC-EV therapy could improve neuron function and reduce infarct volume in a preclinical rodent ischemic stroke model, providing helpful clues for human clinical trials on SC-EVs.

Mesenchymal stromal cell extracellular vesicles for multiple sclerosis in preclinical rodent models: A meta-analysis.

Introduction: Extracellular vesicles (EVs), especially mesenchymal stem (stromal) cell-derived EVs (MSC-EVs), have gained attention as potential novel treatments for multiple sclerosis (MS). However, their effects remain incompletely understood. Thus, the purpose of this meta-analysis was to systematically review the efficacy of MSC-EVs in preclinical rodent models of MS. Methods: We searched PubMed, EMBASE, and the Web of Science databases up to August 2021 for studies that reported the treatment effects of MSC-EVs in rodent MS models. The clinical score was extracted as an outcome. Articles were peer-reviewed by two authors based on the inclusion and exclusion criteria. This meta-analysis was conducted using Stata 15.1 and R. Results: A total of twelve animal studies met the inclusion criteria. In our study, the MSC-EVs had a positive overall effect on the clinical score with a standardized mean difference (SMD) of -2.17 (95% confidence interval (CI)):-3.99 to -0.34, P = 0.01). A significant amount of heterogeneity was observed among the studies. Conclusions: This meta-analysis suggests that transplantation of MSC-EVs in MS rodent models improved functional recovery. Additionally, we identified several critical knowledge gaps, such as insufficient standardized dosage units and uncertainty regarding the optimal dose of MSC-EVs transplantation in MS. These gaps must be addressed before clinical trials can begin with MSC-EVs.

Transplantation of Nasal Olfactory Mucosa Mesenchymal Stem Cells Benefits Alzheimer's Disease.

Alzheimer's disease (AD) is a common neurodegenerative disease that contributes to 60-70% of dementia in elderly people and is currently incurable. Current treatments only relieve the symptoms of AD and slow its progression. Achieving effective neural regeneration to ameliorate cognitive impairment is a major challenge in the treatment of AD. For the first time, we alleviated symptoms of AD in APPswe/PS1dE9 mice (hereafter referred to as AD mice) by transplantation of olfactory mucosa mesenchymal stem cells (OM-MSCs). Our study demonstrated that OM-MSC transplantation promotes amyloid-ß (Aß) clearance, downregulates the inflammatory response, and increases the M2/M1 ratio; OM-MSCs promote the conversion of BV2 (microglia) from M1 to M2 and also Aß clearance in SH-SY5YAPPswe (AD cell model). OM-MSC-transplanted AD mice show improved cognitive learning and locomotive behavior. Our study suggests that OM-MSC transplantation could alleviate the symptoms of AD and promote Aß clearance through immunomodulation, thus demonstrating the great potential and social value of OM-MSC treatment for AD patients.

Effect of Bone Marrow Mesenchymal Stromal Cell Therapies in Rodent Models of Sepsis: A Meta-Analysis.

Background: Multiple preclinical studies have demonstrated that bone-marrow derived mesenchymal stromal (stem) cells [MSC(M)] positively influence the severity of sepsis symptoms and mortality in rodent models. However, this remains an inconclusive finding. Objective: To review the effect of naïve MSC(M) in rodent models of sepsis. Methods: The PubMed, EMBASE, and Web of Science databases were searched up to August 31, 2021. Inclusion criteria according to PICOS criteria were as follows: (1) population: rodents; (2) intervention: unmodified MSC(M); (3) comparison: not specified; (4) primary outcome: the effects of MSC(M) cell therapy on the mortality of rodent models of sepsis and endotoxemia; (5) study: experimental studies. Multiple prespecified subgroup and meta-regression analysis were conducted. Following quality assessment, random effects models were used for this meta-analysis.The inverse variance method of the fixed effects model was used to calculate the pooled odds ratios (ORs) and their 95% confidence intervals (CIs). Results: twenty-four animal studies met the inclusion criteria. Our results revealed an overall OR difference between animals treated with naïve MSC(M) and controls for mortality rate was 0.34(95% confidence interval: 0.27-0.44; P < 0.0001). Significant heterogeneity among studies was observed. Conclusions: The findings of this meta-analysis suggest that naïve MSC(M) therapy decreased mortality in rodent models of sepsis. Additionally, we identified several key knowledge gaps, including the lack of large animal studies and uncertainty regarding the optimal dose of MSC(M) transplantation in sepsis. Before MSC(M) treatment can advance to clinical trials, these knowledge gaps must be addressed.

Hypoxic conditioned promotes the proliferation of human olfactory mucosa mesenchymal stem cells and relevant lncRNA and mRNA analysis.

AIMS: LncRNAs are involved in many biological processes, and hypoxia contributed to the alterations of lncRNAs. Hypoxic preconditioned olfactory mucosa mesenchymal stem cells (OM-MSCs) exerted stronger anti-apoptotic ability in models of disease, but the molecules that controlled different biological characteristics of human OM-MSCs between hypoxic and normoxic conditions were unclear. The present study was aimed to explore the molecules that controlled different biological characteristics of human OM-MSCs between hypoxic and normoxic conditions. MAIN METHODS: LncRNAs and mRNAs expression profiles of human OM-MSCs between hypoxic (3%) and normoxic conditions were analyzed by Next-Generation Sequencing (NGS) analysis, bioinformatics analysis on these data were further performed. Moreover, loss-of function assay was conducted to investigate the impact of hypoxic condition on the proliferation and apoptosis of OM-MSCs. KEY FINDINGS: Through the comparative analysis and bioinformatics analysis, a total of 1741 lncRNAs and 1603 mRNAs were significant differentially expressed in the hypoxia group compared with normoxia group. Enrichment analysis revealed that differentially expressed genes of human OM-MSCs mainly participated in cell cycle regulation, secretin of cytokines and so on. Meanwhile, hypoxic condition significantly promoted proliferation and inhibited apoptosis of human OM-MSCs, following loss-of-function assays confirmed that lncRNA DARS-AS1 were involved in this regulatory process by hypoxic condition. Further prediction of targeted genes and the construction of lncRNA-miRNA-mRNA interaction network enriched the significance regarding the mechanism of DARS-AS1. SIGNIFICANCE: Altogether, these findings provided a new perspective for understanding the molecules expression patterns in hypoxia that contributed to corresponding phenotype alterations of OM-MSCs.

Hypoxia-preconditioned mesenchymal stem cells attenuate microglial pyroptosis after intracerebral hemorrhage.

BACKGROUND: Microglia plays a vital role in neuroinflammation, contributing to the pathogenesis of intracerebral hemorrhage (ICH)-induced brain injury. Mesenchymal stem cells (MSCs) hold great potential for treating ICH. We previously revealed that MSCs ameliorate the microglial pyroptosis caused by an ischemic stroke. However, whether MSCs can modulate microglial pyroptosis after ICH remains unknown. This study aimed to investigate the neuroprotective effects of hypoxia-preconditioned olfactory mucosa MSCs (OM-MSCs) on ICH and the possible mechanisms. METHODS: ICH was induced in mice via administration of collagenase IV. At 6 h post-ICH, 2-4×105 normoxic/hypoxic OM-MSCs or saline were intracerebrally administered. To evaluate the neuroprotective effects, the behavioral outcome, apoptosis, and neuronal injury were measured. Microglia activation and pro-inflammatory cytokines were applied to detect neuroinflammation. Microglial pyroptosis was determined by western blotting, immunofluorescence staining, and transmission electron microscopy (TEM). RESULTS: The two OM-MSC-transplanted groups exhibited significantly improved functional recovery and reduced neuronal injury, especially the hypoxic OM-MSCs group. Hypoxic OM-MSCs attenuated microglial activation as well as the levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). Moreover, we found that hypoxia-preconditioned OM-MSCs ameliorated pyroptosis by diminishing the levels of pyroptosis-associated proteins in peri-hematoma brain tissues, decreasing the expression of the microglial nod-like receptor family protein 3 (NLRP3) and caspase-1, and reducing the membrane pores on microglia post-ICH. CONCLUSIONS: Our study showed that hypoxic preconditioning augments the therapeutic efficacy of OM-MSCs, and hypoxia-preconditioned OM-MSCs alleviate microglial pyroptosis in the ICH model.

Ischemic-hypoxic preconditioning enhances the mitochondrial function recovery of transplanted olfactory mucosa mesenchymal stem cells via miR-181a signaling in ischemic stroke.

Cerebral ischemia/reperfusion injury causes a series of intricate cascade reactions in brain tissue causing apoptosis and proinflammatory programmed cell death known as pyroptosis of nerve cells. The dysfunction of target organelle mitochondria plays a key role in the process of neuronal apoptosis and pyroptosis. Mesenchymal stem cells (MSCs) have been widely used in the experimental or clinical treatment of various ischemic diseases, but the therapeutic efficacy of MSCs on cerebral ischemia-reperfusion injury need to be improved. We successfully cultured olfactory mucosa MSCs (OM-MSCs) to obtain a better source of seed cells. In this way, the therapeutic potential of OM-MSCs transplantation has been evaluated for ischemic stroke using an optimized culture scheme in vitro. Ischemic-hypoxic preconditioned OM-MSCs (IhOM-MSCs) were used to treat a neuron model of oxygen-glucose deprivation/reperfusion and the middle cerebral artery occlusion in rats. These results demonstrated that IhOM-MSCs mediated the upregulation of the downstream target genes GRP78 and Bcl-2 by miR-181a to protect mitochondrial function and inhibit apoptosis and pyroptosis of neurons in the ischemia/reperfusion injury model. Thus, IhOM-MSCs transplantation may be an effective therapy of ischemic stroke in the future.

Hypoxic preconditioning rejuvenates mesenchymal stem cells and enhances neuroprotection following intracerebral hemorrhage via the miR-326-mediated autophagy.

BACKGROUND: Intracerebral hemorrhage (ICH) is a major public health concern, and mesenchymal stem cells (MSCs) hold great potential for treating ICH. However, the quantity and quality of MSCs decline in the cerebral niche, limiting the potential efficacy of MSCs. Hypoxic preconditioning is suggested to enhance the survival of MSCs and augment the therapeutic efficacy of MSCs in ICH. MicroRNAs (miRNAs) are known to mediate cellular senescence. However, the precise mechanism by which miRNAs regulate the senescence of hypoxic MSCs remains to be further studied. In the present study, we evaluated whether hypoxic preconditioning enhances the survival and therapeutic effects of olfactory mucosa MSC (OM-MSC) survival and therapeutic effects in ICH and investigated the mechanisms by which miRNA ameliorates hypoxic OM-MSC senescence. METHODS: In the in vivo model, ICH was induced in mice by administration of collagenase IV. At 24 h post-ICH, 5 × 105 normoxia or hypoxia OM-MSCs or saline was administered intracerebrally. The behavioral outcome, neuronal apoptosis, and OM-MSC survival were evaluated. In the in vitro model, OM-MSCs were exposed to hemin. Cellular senescence was examined by evaluating the expressions of P16INK4A, P21, P53, and by ß-galactosidase staining. Microarray and bioinformatic analyses were performed to investigate the differences in the miRNA expression profiles between the normoxia and hypoxia OM-MSCs. Autophagy was confirmed using the protein expression levels of LC3, P62, and Beclin-1. RESULTS: In the in vivo model, transplanted OM-MSCs with hypoxic preconditioning exhibited increased survival and tissue-protective capability. In the in vitro model, hypoxia preconditioning decreased the senescence of OM-MSCs exposed to hemin. Bioinformatic analysis identified that microRNA-326 (miR-326) expression was significantly increased in the hypoxia OM-MSCs compared with that of normoxia OM-MSCs. Upregulation of miR-326 alleviated normoxia OM-MSC senescence, whereas miR-326 downregulation increased hypoxia OM-MSC senescence. Furthermore, we showed that miR-326 alleviated cellular senescence by upregulating autophagy. Mechanistically, miR-326 promoted the autophagy of OM-MSCs via the PI3K signaling pathway by targeting polypyrimidine tract-binding protein 1 (PTBP1). CONCLUSIONS: Our study shows that hypoxic preconditioning delays OM-MSC senescence and augments the therapeutic efficacy of OM-MSCs in ICH by upregulating the miR-326/PTBP1/PI3K-mediated autophagy.

Olfactory Mucosa Mesenchymal Stem Cells Ameliorate Cerebral Ischemic/Reperfusion Injury Through Modulation of UBIAD1 Expression.

Mesenchymal stem cells (MSCs) have presented a promising neuroprotective effect in cerebral ischemia/reperfusion (I/R). Olfactory mucosa MSCs (OM-MSCs), a novel source of MSCs located in the human nasal cavity, are easy to obtain and situated for autologous transplantation. The present study was designed to evaluate the neuroprotective effects of OM-MSCs on cerebral I/R injury and the possible mechanisms. In the transient middle cerebral artery occlusion (t-MCAO) model, excessive oxidative stress and increased swollen mitochondria were observed in the peri-infarct cortex. Intravenous injection of OM-MSCs ameliorated mitochondrial damage and restored oxidant/antioxidant imbalance. Using the oxygen glucose deprivation/reperfusion (OGD/R) model in vitro, we discovered that the exposure of mouse neuroblastoma N2a cells to OGD/R triggers excessive reactive oxygen species (ROS) generation and induces mitochondrial deterioration with decreased mitochondrial membrane potential and reduces ATP content. OM-MSC transwell coculture attenuated the above perturbations accompanied with increased UbiA prenyltransferase domain-containing 1 (UBIAD1) expression, whereas these protective effects of OM-MSCs were blocked when UBIAD1 was knocked down. UBIAD1-specific small interfering RNA (siRNA) reversed the increased membrane potential and ATP content promoted by OM-MSCs. Additionally, UBIAD1-specific siRNA blocked the oxidant/antioxidant balance treated by OM-MSCs. Overall, our results suggested that OM-MSCs exert neuroprotective effects in cerebral I/R injury by attenuating mitochondrial dysfunction and enhancing antioxidation via upregulation of UBIAD1.

Insight into the proteomic profiling of exosomes secreted by human OM-MSCs reveals a new potential therapy.

Mesenchymal stromal cells (MSCs) have been used for the treatment of neuronal injury and neurodegenerative diseases. Their underlying mechanism may involve increased secretion of paracrine factors, which promotes tissue repair. Presently, exosomes have been regarded as important components of paracrine secretion and paracrine factors. MSC exosomes represent a promising opportunity to develop novel cell-free therapy approaches. In this study, exosomes from nasal olfactory mucosa MSCs (OM-MSCs) were extracted and purified using ultracentrifugation, resulting in exosome diameters of 40-130 nm. Similar to other exosomes, OM-MSC exosomes were CD63- and CD81-positive and calnexin-negative. Functionally, OM-MSC exosomes promoted human brain microvascular endothelial cell (HBMEC) proliferation and migration. The present study analyzed the OM-MSC exosome paracrine proteome. A total of 304 exosome-associated proteins were identified by LC-MS/MS, including plasminogen activator inhibitor 1 (SERPINE 1), insulin-like growth factor binding protein family members (IGFBP 4 and 5), epidermal growth factor receptor (EGFR), neurogenic locus notch homolog protein 2 (NOTCH 2), apolipoprotein E (APOE), and heat shock protein HSP90-beta (HSP90AB1). These molecules are known to be important in neurotrophic, angiogenesis, cell growth, differentiation, apoptosis, and inflammation and are highly correlated with the mechanism of tissue repair and neural restoration. These observations may provide a basis for further evaluation of OM-MSC exosome potential as a novel therapeutic modality.

Olfactory ensheathing cells facilitate neurite sprouting and outgrowth by secreting high levels of hevin.

Transplantation of olfactory ensheathing cells (OECs) has been shown to enhance synapse formation. However, the mechanisms underlying this effect are not completely understood. We performed profiling of the OEC and astrocyte secretomes via a proteomics approach, in case hevin secreted by astrocytes might be involved in the formation of synapses. Semi-quantitative proteomic analysis revealed that 25 proteins were highly expressed, and 22 were weakly expressed in OEC conditioned medium compared with astrocyte conditioned medium. These molecules are highly associated with neural differentiation and regeneration, enzyme regulatory activity, and growth factor binding. The quantification data of clusterin, fibronectin, hevin, insulin-like growth factor binding protein 2 and secreted protein acidic and rich in cysteine were further confirmed by western blotting. Moreover, the addition of hevin in the culture medium improved neurite sprouting and outgrowth of differentiated neural stem cells. The greater expression of hevin in OEC conditioned medium than in astrocyte conditioned medium was associated with a greater capacity of synaptic formation. Thus, our results indicate that soluble factors secreted by OECs provide a permissive environment for nerve repair, and hevin is one of the key molecules facilitating neurite sprouting and outgrowth.