间充质干细胞来源外泌体促心肌梗死血管新生及其机制研究进展

血管新生(angiogenesis)是机体内一个复杂的生理学和病理学过程,是治疗缺血性疾病的重要措施。大量实验研究已表明间充质干细胞(mesenchymal stem cells, MSCs)等干细胞移植可促进心肌梗死后血管新生,近期研究证实这一作用可能主要通过分泌外泌体形式介导。外泌体(exosome)通过传递与血管新生相关微RNA(microRNA, mi RNA)或蛋白质等生物活性物质,调控靶器官中与血管新生相关通路的基因表达,提高内皮细胞在缺血缺氧环境下的存活、迁移、成管能力,促进心肌梗死区域血管新生。通过基因修饰手段增强外泌体介导的心脏修复作用,以及将外泌体与生物活性肽结合形成工程外泌体来靶向缺血心肌治疗,是目前外泌体在心血管领域的热点研究方向。本文结合近年外泌体研究的相关文献,就MSCs来源外泌体促进心肌梗死血管新生的具体机制及现状研究作一综述。     

脂肪间充质干细胞来源外泌体的功能

外泌体是细胞外膜质纳米囊泡,将蛋白质、核酸(DNA和RNA)转运到靶细胞中,介导局部和系统的细胞间通信,从而改变受体细胞的行为.这些小泡在许多生物功能中发挥重要作用,如脂肪合成、免疫调节、神经再生和肿瘤调节等.脂肪间充质干细胞目前被认为是细胞治疗和再生医学领域中一种功能丰富的工具,可产生和分泌多种外泌体,继承细胞的多种...     

间充质干细胞外泌体治疗肺部疾病的研究进展

肺部疾病作为一种呼吸系统常见病,与重工业快速发展、环境污染等密切相关,死亡率极高.近来研究发现间充质干细胞(mesenchymal stem cells,MSCs)能够自主归巢至疾病损伤部位、修复受损组织并参与调节全身炎症和免疫反应,在临床上有较好的应用前景.外泌体是一种细胞外膜囊泡,可能通过调节细胞间通讯参与生物系统...     

人嗅觉黏膜间充质干细胞来源的外泌体促进内皮细胞的血管生成

外泌体作为是细胞旁分泌的重要介质,在促血管形成方面有重要作用。在我们前期研究中,已经成功从嗅黏膜间充质干细胞（olfactory mucosa mesenchymal stem cells,OM-MSCs）分离、鉴定了其外泌体,然而,OM-MSCs源外泌体对血管生成的影响尚不清楚。本研究旨在探讨OM-MSCs来源外泌体对内皮细胞血管生成能力的影响。采用PKH67 荧光标记OM-MSCs源外泌体,与人脑微血管内皮细胞（human brain microvessel endothelial cells, HBMECs) 共培养,观察 OM-MSCs外泌体能否进入 HBMECs。采用CCK-8法、Transwell 迁移实验和小管实验,观察 OM-MSCs外泌体对 HBMECs增殖、迁移及管状结构形成的影响。采用基质胶塞实验及CD31免疫荧光,观察OM-MSCs外泌体在体内对血管生成的影响。上述研究均以等量 PBS 作为对照。结果提示,OM-MSCs外泌体可被HBMECs 摄取。CCK-8 法检测显示,在处理1、2、3、4、5 d各时间点,实验组细胞增殖均优于对照组（1.32±0.14 vs. 0.98±0.04, 1.36±0.14 vs.1.04±0.06, 1.75±0.18 vs.1.33±0.11, 2.16±0.11 vs.1.50±0.19, 2.71±0.11 vs. 1.81±0.20, P<0.01）。Transwell 实验结果显示,实验组跨膜迁移细胞吸光度值较对照组显著增多（1.12±0.05 vs.0.02±0.02, P<0.05）。在体外小管实验中,从节点、交叉点、网眼数、血管分支数和总长度5个方面,实验组均高于空白对照组（374.33±127.74 vs. 193.33±44.79, 104.56±33.07 vs. 54.33±11.65, 20.11±11.20 vs. 7.56±3.64, 81.67±19.07 vs. 57.00±13.02, 11466.22±2781.03 vs. 8544.00±1848.61, P<0.05）;在体内实验中,实验组成血管及CD31阳性率（%）亦显著高于对照组（85.00±5.57 vs.8.00±2.08, P<0.05）。本研究表明：OM-MSCs外泌体可促进 HBMECs 增殖、迁移及管样结构形成,提示OM-MSCs外泌体可促进血管新生。     

人嗅觉黏膜间充质干细胞来源的外泌体促进内皮细胞的血管生成

外泌体作为是细胞旁分泌的重要介质,在促血管形成方面有重要作用。在我们前期研究中,已经成功从嗅黏膜间充质干细胞（olfactory mucosa mesenchymal stem cells,OM-MSCs）分离、鉴定了其外泌体,然而,OM-MSCs源外泌体对血管生成的影响尚不清楚。本研究旨在探讨OM-MSCs来源外泌体对内皮细胞血管生成能力的影响。采用PKH67 荧光标记OM-MSCs源外泌体,与人脑微血管内皮细胞（human brain microvessel endothelial cells, HBMECs) 共培养,观察 OM-MSCs外泌体能否进入 HBMECs。采用CCK-8法、Transwell 迁移实验和小管实验,观察 OM-MSCs外泌体对 HBMECs增殖、迁移及管状结构形成的影响。采用基质胶塞实验及CD31免疫荧光,观察OM-MSCs外泌体在体内对血管生成的影响。上述研究均以等量 PBS 作为对照。结果提示,OM-MSCs外泌体可被HBMECs 摄取。CCK-8 法检测显示,在处理1、2、3、4、5 d各时间点,实验组细胞增殖均优于对照组（1.32±0.14 vs. 0.98±0.04, 1.36±0.14 vs.1.04±0.06, 1.75±0.18 vs.1.33±0.11, 2.16±0.11 vs.1.50±0.19, 2.71±0.11 vs. 1.81±0.20, P<0.01）。Transwell 实验结果显示,实验组跨膜迁移细胞吸光度值较对照组显著增多（1.12±0.05 vs.0.02±0.02, P<0.05）。在体外小管实验中,从节点、交叉点、网眼数、血管分支数和总长度5个方面,实验组均高于空白对照组（374.33±127.74 vs. 193.33±44.79, 104.56±33.07 vs. 54.33±11.65, 20.11±11.20 vs. 7.56±3.64, 81.67±19.07 vs. 57.00±13.02, 11466.22±2781.03 vs. 8544.00±1848.61, P<0.05）;在体内实验中,实验组成血管及CD31阳性率（%）亦显著高于对照组（85.00±5.57 vs.8.00±2.08, P<0.05）。本研究表明：OM-MSCs外泌体可促进 HBMECs 增殖、迁移及管样结构形成,提示OM-MSCs外泌体可促进血管新生。     

间充质干细胞来源外泌体在骨关节炎治疗中的作用

骨关节炎(OA)是最常见的慢性退行性骨关节疾病,目前对骨关节炎的治疗还没有特效疗法。间充质干细胞(MSC)对软骨修复有较好的疗效,间充质干细胞来源外泌体可能在这一治疗过程中发挥重要作用。外泌体是细胞间的通讯载体,能在细胞间传递生物活性脂质、核酸以及蛋白质等生物活性分子对骨关节炎产生一定影响。本文就探讨间充质干细胞来源的外泌体治疗骨关节炎过程中的作用机制与可行性做出综述。     

间充质干细胞来源的外泌体对巨噬细胞的影响

间充质干细胞具有良好的自我更新能力、多向分化潜能和免疫调节性能,目前已应用于多种组织、器官的修复及再生治疗。随着\"无细胞治疗\"研究的不断进展,因为具有和母细胞相似的功能,间充质干细胞来源的外泌体越来越成为治疗各种疾病的研究热点。巨噬细胞是机体固有免疫的重要组成部分,在炎症调控、维持组织内稳定、促进组织的修复与再生方面发挥重要的作用。近来研究发现,巨噬细胞与间充质干细胞的免疫调节特性及其介导的组织再生有密切联系,作为间充质干细胞旁分泌的重要物质,外泌体在调控巨噬细胞的极化中发挥着重要作用。外泌体在与巨噬细胞产生作用后,可以促使巨噬细胞由M1型向M2型转化,促进巨噬细胞M2型活化,从而发挥其生物学作用。本文将对间充质干细胞来源的外泌体的分离、生物学特性、对巨噬细胞的影响及其在疾病治疗中的应用前景进行综述。     

间充质干细胞外泌体在阿尔兹海默症治疗中的研究进展

阿尔兹海默症（AD）是一种病理机制复杂,以进行性认知功能障碍为主的中枢神经系统疾病,目前仍缺乏有效的治疗方法。多项研究结果显示,间充质干细胞（MSCs）外泌体能够促进抗炎、调节免疫功能、加强Aβ降解、促进神经细胞轴突生长等,能很好地针对AD的核心病理机制发挥效果从而达到治疗效果。本文主要介绍MSCs外泌体在各项AD病理机制治疗中的研究进展。     

间充质干细胞外泌体对海马星形胶质细胞活化的抑制作用研究

目的 探讨间充质干细胞外泌体(MSC-Exo)对海马星形胶质细胞活化的抑制作用.方法 实验通过超速离心法提取脐带MSC-Exo,并使用PKH-26染料标记;MSC-Exo预处理原代海马星形胶质细胞后使用脂多糖(LPS)诱导细胞活化,并分为对照组、LPS组和LPS+MSC-Exo组,进而行免疫细胞化学检测胶质纤维酸性蛋白...     

干细胞来源外泌体在骨关节炎的病理生理发展进程的作用和诊断治疗的研究进展

在过去的十几年中,随着外泌体(从细胞释放的囊泡)的发现,鉴定和研究的深入,除了诸多研究发现病理情况下细胞分泌的外泌体对于骨关节炎疾病的病理生理进程具有重要作用之外,还发现了外泌体作为良好的生物信号传递因子,具有更加强大的软骨再生修复的应用潜能。合理调控和抑制"致病的"外泌体,合理利用"优良的"外泌体,对于骨关节炎的病理进程的控制和疾病的治疗都具有重要的意义。本文主要综述在骨关节炎疾病中外泌体的主要致病作用和机制,以及使用干细胞来源外泌体进行骨关节炎疾病治疗的研究进展。     

间充质干细胞源性外泌体miRNAs参与损伤心血管细胞修复的研究进展

外泌体是由各种组织和细胞所释放的细胞外囊泡,在细胞之间充当各种分子(包括蛋白质,脂质和RNA)的载体,从而调节或干预特定的生理过程.MiRNAs是一类小的非编码RNA,能够靶向多个mRNA并诱导其降解或翻译抑制以调控相关基因表达.目前对外泌体内微小RNA(miR-NAs)的研究最为广泛.间充质干细胞(MSCs)是一种多...     

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.     

K562 cell-derived exosomes suppress the adhesive function of bone marrow mesenchymal stem cells via delivery of miR-711

A failure of bone marrow mesenchymal stem cells (BM-MSCs) to adhere to hematopoietic cells is an essential cause of the progression of chronic myelogenous leukemia and is also a cause of failure of bone marrow (BM) transplantation, but the exact mechanisms of this have not been fully elucidated. Recent studies have indicated that microRNAs (miRNAs) are contained in leukemia-derived exosomes and are involved in modulating the BM microenvironment. In this study, we found that K562 cell-derived exosomes transfer miR-711 to BM-MSCs and suppress the adhesive function of BM-MSCs. Using qRT-PCR, we also confirmed a significantly higher level of miR-711 in exosomes derived from K562 cells than in exosomes derived from parental cells. The BM-MSCs co-cultured with exosomes derived from K562 cells showed a lower adhesion rate than did controls. We further demonstrated that exosomal transfer of miR-711 induced decreased adhesive abilities by inhibiting expression of adhesion molecule CD44 in BM-MSCs. In conclusion, our study reveals that K562 cell-derived exosomal miR-711 can be transferred to BM-MSCs and weaken adhesive abilities by silencing the expression of the adhesion molecule CD44.     

Activation of NRG1-ERBB4 signaling potentiates mesenchymal stem cell-mediated myocardial repairs following myocardial infarction

Mesenchymal stem cell (MSC) transplantation has achieved only modest success in the treatment of ischemic heart disease owing to poor cell viability in the diseased microenvironment. Activation of the NRG1 (neuregulin1)-ERBB4 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4) signaling pathway has been shown to stimulate mature cardiomyocyte cell cycle re-entry and cell division. In this connection, we aimed to determine whether overexpression of ERBB4 in MSCs can enhance their cardio-protective effects following myocardial infarction. NRG1, MSCs or MSC-ERBB4 (MSC with ERBB4 overexpression), were transplanted into mice following myocardial infarction. Superior to that of MSCs and solely NRG1, MSC-ERBB4 transplantation significantly preserved heart functions accompanied with reduced infarct size, enhanced cardiomyocyte division and less apoptosis during early phase of infarction. The transduction of ERBB4 into MSCs indeed increased cell mobility and apoptotic resistance under hypoxic and glucose-deprived conditions via a PI3K/Akt signaling pathway in the presence of NRG1. Unexpectedly, introduction of ERBB4 into MSC in turn potentiates NRG1 synthesis and secretion, thus forming a novel NRG1-ERBB4-NRG1 autocrine loop. Conditioned medium of MSC-ERBB4 containing elevated NRG1, promoted cardiomyocyte growth and division, whereas neutralization of NRG1 blunted this proliferation. These findings collectively suggest that ERBB4 overexpression potentiates MSC survival in the infarcted heart, enhances NRG1 generation to restore declining NRG1 in the infarcted region and stimulates cardiomyocyte division. ERBB4 has an important role in MSC-mediated myocardial repairs.Although mesenchymal stem cell (MSC)-based cell transplantation is a promising and novel approach for cardiac repair following myocardial infarction (MI) that involves paracrine factors and cardiovascular differentiation,^{1, 2, 3, 4} the poor survival and engraftment of transplanted stem cells within the ischemic myocardium remain major limitations to this process. Numerous strategies have been used to improve MSC-based therapeutic potential, among which genetic modification has drawn considerable attention. Introducing genes into MSCs to enhance cell viability, mobility and angiogenesis has been explored.^{5, 6, 7, 8} For example, overexpression of the anti-apoptotic factor Bcl-2 (B-cell lymphoma 2) in MSCs enhances survival capacity and improves cardiac performance during MI following transplantation.⁹ Similarly, MSCs with IGF1 (insulin-like growth factor 1) overexpression induce stem cell mobilization and increase angiomyogenesis that subsequently contribute to myocardial repair.¹⁰ Nevertheless the effects of these genetically engineered MSCs on adult mature cardiomyocyte regeneration are unclear. Identification of novel genes or pathways that both ameliorate MSC properties and initiate endogenous cardiomyocyte regeneration in situ will thus provide crucial benefits.Following examination of adult hearts, Bersell et al.¹¹ recently revealed that NRG1 (neuregulin1) can stimulate mononuclear mature cardiomyocytes to re-enter the cell cycle and DNA duplicate status through the NRG1-ERBB signaling pathway. Strategic targeting of the signaling complex of NRG1 and its tyrosine kinase receptors, ERBB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2) and ERBB4 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4) has demonstrated positive outcomes following heart failure in animal models.^{12, 13, 14} NRG1, a member of the epidermal growth factor family, has been comprehensively documented as an essential paracrine regulator of cell–cell communication through activation of its ERBB tyrosine kinase receptors, and is indispensable in heart development and adult cardiac physiology.^{11, 15, 16, 17, 18, 19} The recombinant human NRG1 has been used in clinical trials to treat chronic heart failure in China and Australia (ClinicalTrials.gov identifiers {"type":"clinical-trial","attrs":{"text":"NCT01131637","term_id":"NCT01131637"}}NCT01131637, {"type":"clinical-trial","attrs":{"text":"NCT01258387","term_id":"NCT01258387"}}NCT01258387), in which it proved to enhance heart function and reverse remodeling.^{20, 21} ERBB2 acts as a non-ligand binding, pre-activated co-receptor that needs to heterodimerize with ERBB4 and forms an ERBB2/ERBB4 complex upon activation by NRG1. In contrast, ERBB4 possesses the capability of homodimerization and activates PI3K/Akt (v-akt murine thymoma viral oncogene homolog 1) signaling that facilitates cell survival. Although this crucial discovery suggests the potential of using NRG1 as a therapeutic agent for cardiac repair, numerous challenges must be addressed before it can become a reality. First, the circulating half-life of NRG1 is extremely short (~30 min),¹⁴ thus the interval during which it can be delivered is limited. Attempts have been made to prolong the NRG1 half-life by using a controlled delivery²² or by combining it with adipose-derived stem cell transplantation.²³ Second, a relatively small population (10%) of cardiomyocytes in the adult heart are mononucleated and although systematic administration of NRG1 initiates cytokinesis in vivo, it only corresponds to ~0.3% of the mononuclear cardiomyocytes present following MI.¹¹ Third, the receptor presenting on the targeting cells has a vital role in managing whether the cells respond to NRG1 stimuli or not. These sub-optimal results may attribute to the low penetration of NRG1 or in the ischemic myocardium that consequently limits its therapeutic efficiency.Both MSC therapy and targeting the NRG1-ERBB complex hold great potential for treating cardiac diseases. Our hypothesis is therefore to combine these advantages by engineering MSCs and studying whether and how these cells can confer cardiac protection following transplantation into infarcted hearts. To this end, we first screened expression of ERBB receptors in mouse MSCs and determined that NRG1 and ERBB2, but not ERBB4, were expressed in MSCs. As ERBB2 is incompetent to bind NRG1, transplanting unmodified MSCs that lack ERBB4 cannot activate NRG1-ERBB signaling, and thus stultifies the existing NRG1 in the infarcted myocardium.²⁴ We introduced ERBB4 expression into MSCs (MSC-ERBB4) and transplanted them into MI model. MSC-ERBB4 not only significantly preserved heart functions accompanied with reduced infarct size and enhanced cardiomyocyte division, but also improved MSC survival and created an autocrine loop in the NRG1-ERBB4-NRG1 signaling pathway. These results suggest that engineering ERBB4 in MSCs could be an novel strategy to benefit both MSCs and cardiomyocytes for enhancing efficacy of MSC-based transplantation in heart infarction.     

ATL-derived exosomes modulate mesenchymal stem cells: potential role in leukemia progression

Background

Exosomes are membrane nano-vesicles secreted by a multitude of cells that harbor biological constituents such as proteins, lipids, mRNA and microRNA. Exosomes can potentially transfer their cargo to other cells, implicating them in many patho-physiological processes. Mesenchymal stem cells (MSCs), residents of the bone marrow and metastatic niches, potentially interact with cancer cells and/or their derived exosomes. In this study, we investigated whether exosomes derived from adult T-cell leukemia/lymphoma (ATL) cells act as intercellular messengers delivering leukemia-related genes that modulate the properties of human MSCs in favor of leukemia. We hypothesized that the cargo of ATL-derived exosomes is transferred to MSCs and alter their functional behavior to support the establishment of the appropriate microenvironment for leukemia.

Results

We showed that both ATL cells (C81 and HuT-102) and patient-derived cells released Tax-containing exosomes. The cargo of HuT-102-derived exosomes consisted of miR-21, miR-155 and vascular endothelial growth factor. We demonstrated that HuT-102-derived exosomes not only deliver Tax to recipient MSCs, but also induce NF-κB activation leading to a change in cellular morphology, increase in proliferation and the induction of gene expression of migration and angiogenic markers.

Conclusions

This study demonstrates that ATL-derived exosomes deliver Tax and other leukemia-related genes to MSCs and alter their properties to presumably create a more conducive milieu for leukemia. These findings highlight the contribution of leukemia-derived exosomes in cellular transformation and their potential value as biomarkers and targets in therapeutic strategies.

     

Imaging long-term fate of intramyocardially implanted mesenchymal stem cells in a porcine myocardial infarction model

The long-term fate of stem cells after intramyocardial delivery is unknown. We used noninvasive, repetitive PET/CT imaging with [(18)F]FEAU to monitor the long-term (up to 5 months) spatial-temporal dynamics of MSCs retrovirally transduced with the sr39HSV1-tk gene (sr39HSV1-tk-MSC) and implanted intramyocardially in pigs with induced acute myocardial infarction. Repetitive [(18)F]FEAU PET/CT revealed a biphasic pattern of sr39HSV1-tk-MSC dynamics; cell proliferation peaked at 33-35 days after injection, in periinfarct regions and the major cardiac lymphatic vessels and lymph nodes. The sr39HSV1-tk-MSC-associated [(18)F]FEAU signals gradually decreased thereafter. Cardiac lymphography studies using PG-Gd-NIRF813 contrast for MRI and near-infrared fluorescence imaging showed rapid clearance of the contrast from the site of intramyocardial injection through the subepicardial lymphatic network into the lymphatic vessels and periaortic lymph nodes. Immunohistochemical analysis of cardiac tissue obtained at 35 and 150 days demonstrated several types of sr39HSV1-tk expressing cells, including fibro-myoblasts, lymphovascular cells, and microvascular and arterial endothelium. In summary, this study demonstrated the feasibility and sensitivity of [(18)F]FEAU PET/CT imaging for long-term, in-vivo monitoring (up to 5 months) of the fate of intramyocardially injected sr39HSV1-tk-MSC cells. Intramyocardially transplanted MSCs appear to integrate into the lymphatic endothelium and may help improve myocardial lymphatic system function after MI.