WHOM will be doing the same research for stroke? And then with human testing? With NO leadership and NO strategy NOTHING WILL BE DONE!
Exosome Derived from Human Neural Stem Cells Improves Motor Activity and Neurogenesis in a Traumatic Brain Injury Model
Mahsa Abedi
,1,2 Mehrdad Hajinejad,3,4 Fereshteh Atabi,1 and Sajad Sahab-Negah5,61Department of Biochemistry and Biophysics, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
2Shefa Neuroscience Research Center, Khatam Alanbia Hospital, Tehran, Iran
3Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
4Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran
5Neuroscience Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
6Department of Neuroscience, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Abstract
Traumatic brain injury (TBI) is a leading cause of mortality and long-lasting disability globally. Although novel treatment options have been investigated, no effective therapeutic opportunities for TBI exist. Accumulating studies demonstrated that the paracrine mechanisms of stem cells may allow them to orchestrate regenerative processes after TBI. So far, very little attention has been paid to the beneficial effects of human neural stem cells (hNSCs) in comparison to their exosomes as a paracrine mechanism. This study is aimed at comparing the effect of hNSCs with their exosomes in a TBI model. For in vitro assessments, we cultured hNSCs using the neurosphere method and isolated hNSC-derived exosomes from culture supernatants. For in vivo experiments, male rats were divided into three groups (
hNSCs after TBI; and exosome group: rats received a single intralesional injection of 63 μg protein of hNSC-derived exosomes after TBI. Neurological assessments, neuroinflammation, and neurogenesis were performed at the predetermined time points after TBI. Our results indicated that the administration of exosomes improved the neurobehavioral performance measured by the modified neurological severity score (mNSS) on day 28 after TBI. Furthermore, exosomes inhibited the expression of reactive astrocytes as a key regulator of neuroinflammation marked by GFAP at the protein level, while enhancing the expression of Doublecortin (DCX) as a neurogenesis marker at the mRNA level. On the other hand, we observed that the expression of stemness markers (SOX2 and Nestin) was elevated in the hNSC group compared to the exosome and TBI groups. To sum up, our results demonstrated that the superior effects of exosomes versus parent hNSCs could be mediated by improving mNSS score and increasing DCX in TBI. Considerably, more work will need to be done to determine the beneficial effects of exosomes versus parent cells in the context of TBI.
1. Introduction
Traumatic brain injury (TBI) is caused by a mechanical force on the brain tissue [1]. TBI severity can be classified into severe, moderate, and mild by scores on the Glasgow Coma Scale that can cause substantial neurological disabilities and mental distress [2]. TBI has been estimated to create an alarming rate in the United States with 10 million cases annually and become the third leading cause of death worldwide [3]. TBI does not have a single pathophysiological appearance; it is a multimodal complex disease process in which primary and secondary injuries induce numerous pathological changes to the brain parenchyma [1, 2]. Proinflammatory cytokines secreted by glial cells (i.e., astrocytes and microglia) play important roles in the pathogenesis of TBI [4]. Glutamate toxicity and oxidative stress are other prominent molecular mechanisms that induce cell death and increase the severity of TBI [1, 5]. Despite global efforts to find out effective treatment, there is no cure treatment for TBI [6]. During the last decades, stem cell treatment strategies have shown promising results in experimental and some clinical studies [7]. In recent years, different types of stem cells, such as mesenchymal stem cells (MSCs), hematopoietic stem cells, umbilical cord stem cells, and neural stem cells (NSCs), have been investigated in the course of TBI [6]. For example, transplantation of NSCs promoted functional recovery after a TBI model by increasing synaptic density [8]. Recently, our team showed that NSC therapy in conjunction with nanocurcumin increased recovery from TBI by decreasing astrogliosis and its downstream neuroinflammatory pathways [9]. However, several challenges remain to be overcome before stem cell therapy can become a reality for patients [10, 11]. For example, immune responses after transplantation, oncogenic properties, low neuronal differentiation capacity, and low cell engraftment should be carefully assessed [12]. Recently, bystander effects of stem cells (i.e., paracrine mechanisms) have been introduced as an alternative approach to stem cell therapy [13, 14]. Among different bystander mechanisms, a new mechanism for intercellular communication has emerged which involves the intercellular transfer of exosomes [15]. Up to now, numerous studies have been done on the beneficial effects of exosomes derived from MSCs in neurological disorders. They presented reasonable explanations of why exosomes are valuable therapeutic agents for TBI [16, 17]. Exosomes are nanosized extracellular vesicles (30-100 nm) that carry cell-specific cargos of proteins, lipids, and RNA (mRNA, noncoding RNA, etc.) [18]. Exosomes can affect the physiological function of target cells by regulating gene expression or protein synthesis [17]. It should be noted that there has been little discussion about the beneficial effects of exosomes derived from human neural stem cells (hNSCs) in the course of TBI. Hence, we hypothesized that exosomes derived from hNSCs could be advantageous for TBI and thus offer a better way for treatments. Therefore, the present study primarily investigated whether hNSC-derived exosomes could improve functional recovery and pathological changes after TBI. More essentially, we compared the beneficial effects of hNSC-derived exosomes with hNSCs in an experimental brain injury model for the first time.
2. Materials and Methods
2.1. In Vitro Assessments
2.1.1. Culture of hNSCs
hNSCs were purchased from the Biobank of Neuroscience Department of Mashhad University of Medical Sciences. hNSCs were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (Gibco, Germany) containing 1.5% fetal bovine serum (FBS) (Gibco, Germany), 0.5% penicillin-streptomycin (Pen/Strep) (Gibco, Germany), 0.5% L-glutamine (Gibco, Germany), 0.5% B27 (Gibco, Germany), 0.25% N2 (Gibco, Germany), and 20 ng/mL epidermal growth factor (EGF) (Sigma, Germany).
The hNSCs were characterized with ICC staining against nestin and SOX2 proteins as a neural stem cell marker.
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