Pre‐treatments enhance the therapeutic effects of mesenchymal stem cells in liver diseases

Liver diseases caused by viral infection, alcohol abuse and metabolic disorders can progress to end‐stage liver failure, liver cirrhosis and liver cancer, which are a growing cause of death worldwide. Although liver transplantation and hepatocyte transplantation are useful strategies to promote liver regeneration, they are limited by scarce sources of organs and hepatocytes. Mesenchymal stem cells (MSCs) restore liver injury after hepatogenic differentiation and exert immunomodulatory, anti‐inflammatory, antifibrotic, antioxidative stress and antiapoptotic effects on liver cells in vivo. After isolation and culture in vitro, MSCs are faced with nutrient and oxygen deprivation, and external growth factors maintain MSC capacities for further applications. In addition, MSCs are placed in a harsh microenvironment, and anoikis and inflammation after transplantation in vivo significantly decrease their regenerative capacity. Pre‐treatment with chemical agents, hypoxia, an inflammatory microenvironment and gene modification can protect MSCs against injury, and pre‐treated MSCs show improved hepatogenic differentiation, homing capacity, survival and paracrine effects in vitro and in vivo in regard to attenuating liver injury. In this review, we mainly focus on pre‐treatments and the underlying mechanisms for improving the therapeutic effects of MSCs in various liver diseases. Thus, we provide evidence for the development of MSC‐based cell therapy to prevent acute or chronic liver injury. Mesenchymal stem cells have potential as a therapeutic to prolong the survival of patients with end‐stage liver diseases in the near future.     

间充质干细胞特性与应用前景

间充质干细胞是中胚层发育的早期细胞,具备干细胞的基本特性。在发育的不同阶段和特定环境条件下,间充质干细胞可向骨、软骨、肌肉、神经、血管及血液细胞等多种方向分化。在成体的很多器官和组织中也存在着间充质干细胞,以备修复和再生所用。间充质干细胞易于体外培养,扩增迅速,可以分化为多种细胞,为干细胞生物工程提供了一个很好的种子细胞。在明确间充质干细胞生物学特性和分化的机制后,可在体外和体内将其定向诱导分化为多种细胞。间充质干细胞具有巨大的临床应用价值和科学研究价值。     

Effect of mesenchymal stem cell-derived exosomes on the induction of mouse tolerogenic dendritic cells

Dendritic cells (DCs) orchestrate innate inflammatory responses and adaptive immunity through T-cell activation via direct cell–cell interactions and/or cytokine production. Tolerogenic DCs (tolDCs) help maintain immunological tolerance through the induction of T-cell unresponsiveness or apoptosis, and generation of regulatory T cells. Mesenchymal stromal cells (MSCs) are adult multipotent cells located within the stroma of bone marrow (BM), but they can be isolated from virtually all organs. Extracellular vesicles and exosomes are released from inflammatory cells and act as messengers enabling communication between cells. To investigate the effects of MSC-derived exosomes on the induction of mouse tolDCs, murine adipose-derived MSCs were isolated from C57BL/6 mice and exosomes isolated by ExoQuick-TC kits. BM-derived DCs (BMDCs) were prepared and cocultured with MSCs-derived exosomes (100 μg/ml) for 72 hr. Mature BMDCs were derived by adding lipopolysaccharide (LPS; 0.1μg/ml) at Day 8 for 24 hr. The study groups were divided into (a) immature DC (iDC, Ctrl), (b) iDC + exosome (Exo), (c) iDC + LPS (LPS), and (d) iDC + exosome + LPS (EXO + LPS). Expression of CD11c, CD83, CD86, CD40, and MHCII on DCs was analyzed at Day 9. DC proliferation was assessed by coculture with carboxyfluorescein succinimidyl ester-labeled BALB/C-derived splenocytes p. Interleukin-6 (IL-6), IL-10, and transforming growth factor-β (TGF-β) release were measured by enzyme-linked immunosorbent assay. MSC-derived exosomes decrease DC surface marker expression in cells treated with LPS, compared with control cells ( ≤ .05). MSC-derived exosomes decrease IL-6 release but augment IL-10 and TGF-β release (p ≤ .05). Lymphocyte proliferation was decreased (p ≤ .05) in the presence of DCs treated with MSC-derived exosomes. CMSC-derived exosomes suppress the maturation of BMDCs, suggesting that they may be important modulators of DC-induced immune responses.     

脐带间充质干细胞移植对慢性试验性肝损伤的治疗作用

目的观察脐带间充质干细胞（UC-MSC）对慢性实验性肝损伤的治疗作用并探讨其分子生物学机理。方法 50只7周龄的NOD/SCID小鼠注射四氯化碳（CCL4）制备慢性肝损伤模型后,应用随机数字表的方法随机将实验小鼠随机分成2组：模型组（25只）和UC-MSC移植组（25只）。UC-MSC移植组通过尾静脉注射移植1×106 UC-MSC,模型组注射同样体积的PBS。分别于移植后1、2、3和4周收集肝组织,应用免疫组织化学,RT-PCR和Western blot的方法分析细胞移植前后肝组织的病理生理学特征的变化。采用t检验和方差分析进行统计学分析。结果 UC-MSC移植治疗后肝组织表达人肝细胞特异性AFP,Alb,和内皮细胞特异性CD31,Flk-1。细胞移植4周后v WF标记的血管密度明显增加,同时伴有部分的肝功能改善,谷丙转氨酶（ALT）水平从（55.71±11.33）U/L减至（36.75±12.80）U/L（P〈0.05）。此外,本研究结果表明UC-MSC分泌几种重要的生长因子HGF,FGF-2,VEGF,和VEGF受体通过旁分泌的途径发挥肝组织修复的功能。结论在CCL4诱导的慢性肝损伤模型肝组织,人UC-MSC可以分化成肝细胞样细胞和内皮细胞样细胞,同时旁分泌多种细胞生长因子修复损伤的肝细胞,并伴有肝功能的改善。认为UCMSC移植或许成为将来肝脏损伤疾病一个重要的治疗选择。     

骨髓间充质干细胞外泌体中miRNA在视网膜新生血管形成中的作用及机制

视网膜血管疾病如早产儿视网膜病变、糖尿病视网膜病变和视网膜静脉阻塞等以异常增生的视网膜新生血管为主要病理表现。骨髓间充质干细胞来源外泌体通过旁分泌作用传递生物活性分子介导细胞间的物质与信息交换。其中,miRNA等内容物在传递信息中起关键作用,可调控缺血缺氧环境下内皮细胞的增殖、管腔形成和新生血管的形成。并且能够通过血视网膜屏障而不引起免疫、炎症反应,在眼科疾病治疗中极具潜力。本文总结骨髓间充质干细胞衍生外泌体中miRNA在视网膜新生血管形成中的作用和可能的作用机制,以期为外泌体在眼科疾病诊治中的应用拓宽新思路。     

Interleukin‐6 downregulation with mesenchymal stem cell differentiation results in loss of immunoprivilege

Allogeneic mesenchymal stem cell (MSC) transplantation improves cardiac function, but cellular differentiation results in loss of immunoprivilege and rejection. To explore the mechanism involved in this immune rejection, we investigated the influence of interleukin‐6 (IL‐6), a factor secreted by MSCs, on immune privilege after myogenic, endothelial and smooth muscle cell differentiation induced by 5‐azacytidine, VEGF, and transforming growth factor‐β (TGF‐β), respectively. Both RT‐PCR and ELISA showed that myogenic differentiation of MSCs was associated with significant downregulation of IL‐6 expression (P < 0.01), which was also observed following endothelial (P < 0.01) and smooth muscle cell differentiation (P < 0.05), indicating that IL‐6 downregulation was dependent on differentiation but not cell phenotype. Flow cytometry demonstrated that IL‐6 downregulation as a result of myogenic differentiation was associated with increased leucocyte‐mediated cell death in an allogeneic leucocyte co‐culture study (P < 0.01). The allogeneic reactivity associated with IL‐6 downregulation was also observed following MSC differentiation to endothelial and smooth muscle cells (P < 0.01), demonstrating that leucocyte‐mediated cytotoxicity was also dependent on differentiation but not cell phenotype. Restoration of IL‐6 partially rescued the differentiated cells from leucocyte‐mediated cell death. These findings suggest that rejection of allogeneic MSCs after implantation may be because of a reduction in cellular IL‐6 levels, and restoration of IL‐6 may be a new target to retain MSC immunoprivilege.     

间充质干细胞的来源及其对肝脏损伤及修复的研究进展

全球终末期肝病、肝衰竭的发病率和死亡率逐年升高,且目前肝移植是唯一疗效确切的治疗选择,但是,肝移植的使用受到肝源供体严重不足,长期存活率低,医疗费用昂贵等缺点使得原位肝移植的应用受限,绝大多数患者无法受益。为了克服肝脏器官短缺,干细胞替代治疗策略逐渐成为另一个肝病治疗的重要选择,干细胞治疗,特别是间充质干细胞（MSC）提供了一个新的肝病治疗选择。MSC是一群贴壁生长的成纤维细胞样细胞,由于MSC能够分化为多种类型的细胞,能够产生多种的细胞因子和生长因子,具有造血支持和免疫调节和抗炎功能,MSC被认为在再生医学领域具有重大的科学和实用价值。另外,由于MSC应用于治疗实验性肝损伤能明显提高动物存活率,明显改善肝功能。此外,一些临床前研究和临床研究也表明MSC对肝损伤性疾病具有显著地疗效。因此MSC在损伤性和退行性肝脏疾病的治疗具有广阔的应用前景。本文综述了MSC在肝损伤疾病治疗应用的进展,并对MSC在肝病治疗中的应用前景进行了展望。     

Strategies to improve the efficiency of mesenchymal stem cell transplantation for reversal of liver fibrosis

End‐stage liver fibrosis frequently progresses to portal vein thrombosis, formation of oesophageal varices, hepatic encephalopathy, ascites, hepatocellular carcinoma and liver failure. Mesenchymal stem cells (MSCs), when transplanted in vivo, migrate into fibrogenic livers and then differentiate into hepatocyte‐like cells or fuse with hepatocytes to protect liver function. Moreover, they can produce various growth factors and cytokines with anti‐inflammatory effects to reverse the fibrotic state of the liver. In addition, only a small number of MSCs migrate to the injured tissue after cell transplantation; consequently, multiple studies have investigated effective strategies to improve the survival rate and activity of MSCs for the treatment of liver fibrosis. In this review, we intend to arrange and analyse the current evidence related to MSC transplantation in liver fibrosis, to summarize the detailed mechanisms of MSC transplantation for the reversal of liver fibrosis and to discuss new strategies for this treatment. Finally, and most importantly, we will identify the current problems with MSC‐based therapies to repair liver fibrosis that must be addressed in order to develop safer and more effective routes for MSC transplantation. In this way, it will soon be possible to significantly improve the therapeutic effects of MSC transplantation for liver regeneration, as well as enhance the quality of life and prolong the survival time of patients with liver fibrosis.     

Circulating monocytes accelerate acute liver failure by IL‐6 secretion in monkey

Acute liver failure (ALF) is associated with high mortality, and a poor understanding of the underlying pathophysiology has resulted in a lack of effective treatments so far. Here, using an amatoxin‐induced rhesus monkey model of ALF, we panoramically revealed the cellular and molecular events that lead to the development of ALF. The challenged monkeys with toxins underwent a typical course of ALF including severe hepatic injury, systemic inflammation and eventual death. Adaptive immune was not noticeably disturbed throughout the progress of ALF. A systematic examination of serum factors and cytokines revealed that IL‐6 increase was the most rapid and drastic. Interestingly, we found that IL‐6 was mainly produced by circulating monocytes. Furthermore, ablation of monocyte‐derived IL‐6 in mice decreased liver injury and systemic inflammation following chemical injection. Our findings reveal a critical role of circulating monocytes in initiating and accelerating ALF, indicating a potential therapeutic target in clinical treatment for ALF.     

Human umbilical cord mesenchymal stromal cells rescue mice from acetaminophen-induced acute liver failure

Background aimsAcute liver failure (ALF), a life-threatening disease characterized by the sudden loss of hepatic function, can occur after an accidental or intentional acetaminophen overdose.MethodsWith the use of an ALF mouse model, we examined both the preventive and therapeutic potential of intravenously administered human umbilical cord–derived mesenchymal stromal cells (hUCMSCs). Primary hUCMSCs were purified from freshly collected full-term umbilical cords and intravenously transplanted into BALB/c mice either before and after ALF induced by acetaminophen intoxication. We found that hUCMSCs significantly improved survival rates and relative liver weight of mice in both pre-ALF and post-ALF animals. Correspondingly, serum levels of markers that reflect hepatic injury (ie, aspartate aminotransferase, alanine aminotransferase and total bilirubin) were significantly attenuated in the group receiving hUCMSC therapy.ResultsMechanistically, we found that the protective potential of intravenously administered hUCMSCs was mediated by paracrine pathways that involved antioxidants (glutathione, superoxide dismutase), the reduction of inflammatory agents (tumor necrosis factor-α, interleukin-6) and elevated serum levels of hepatocyte growth factor.ConclusionsThrough these paracrine effects, intravenously administered hUCMSCs reduced hepatic necrosis/apoptosis and enhanced liver regeneration. Thus, our data demonstrate that intravenously administered hUCMSCs may be useful in the prevention or treatment of acetaminophen-induced ALF.     

骨髓间充质干细胞移植对急性肝功能衰竭大鼠肝组织miRNA-155和TNF-α表达的影响

目的探讨骨髓间充质干细胞（BMSCs）移植对急性肝功能衰竭（ALF）大鼠肝组织中miRNA-155和TNF-α表达的影响,以及与BMSCs疗效间的关系。方法将SD大鼠随机分为健康对照组、ALF组、BMSCs治疗组和BMSCs预防组,其中ALF组予以900 mg/kg D-GalN＋10μg/kg脂多糖腹腔注射建立模型;BMSCs治疗组在900 mg/kg D-GalN＋10μg/kg脂多糖腹腔注射后2 h,予以尾静脉注射BMSCs 5.0×10^6;BMSCs预防组在900 mg/kg D-GalN＋10μg/kg脂多糖腹腔注射前予以尾静脉注射BMSCs 5.0×10^6;健康对照组予以0.9﹪氯化钠溶液1 ml腹腔注射。给药7 h后每组处死大鼠,检测大鼠血清ALT和AST,ELISA法检测TNF-α水平,实时定量PCR检测肝组织miRNA-155、TNF-αmRNA。各组间肝功指标差异采用方差分析,同时观察每组大鼠的24 h生存率,并用卡方检验比较各组生存率的差异。结果 D-GalN/脂多糖诱导7 h后,与ALF组相比,BMSCs预防和BMSCs治疗组大鼠ALT、AST、TNF-α水平均有所降低（P〈0.01）;同时两组肝组织TNF-αmRNA和miRNA-155表达水平均有下调（P〈0.01）;但两组间相比较差异无统计学意义。ALF组大鼠肝组织miRNA-155上调和TNF-αmRNA诱导呈正相关（r=0.734,P=0.001）。BMSCs预防组和BMSCs治疗组miRNA-155和TNF-αmRNA的部分逆转亦呈正相关（r值分别为0.687和0.590,P值分别为0.004和0.006）。给药后24 h,健康对照组、ALF组、BMSCs治疗组和BMSCs预防组大鼠死亡率组间比较差异有统计学意义（c2=19.078,P〈0.01）。结论在BMSCs干预大鼠ALF发病过程中,可以部分逆转上调的肝组织miRNA-155和TNF-α,且存在协同性,提示BMSCs治疗ALF可能通过对肝组织miRNA-155和TNF-α的调控发生作用。     

间充质干细胞在脂肪性供肝肝移植中的应用与展望

骨髓间充质干细胞（BMSC）是一种具有多向分化能力且能无限增殖的间质细胞,在肝脏疾病的作用已经得到证实。肝移植是目前治疗终末期肝病最为有效的方法,随着脂肪性供肝等边缘性供肝的增加,如何使边缘性供肝更好的发挥功能,成为了新的研究热点。研究BMSC对脂肪性供肝肝移植术后肝脏功能恢复的影响将为其在临床上的应用提供依据。     

Advances in mesenchymal stem cell transplantation for the treatment of osteoporosis

Osteoporosis is a systemic metabolic bone disease with characteristics of bone loss and microstructural degeneration. The personal and societal costs of osteoporosis are increasing year by year as the ageing of population, posing challenges to public health care. Homing disorders, impaired capability of osteogenic differentiation, senescence of mesenchymal stem cells (MSCs), an imbalanced microenvironment, and disordered immunoregulation play important roles during the pathogenesis of osteoporosis. The MSC transplantation promises to increase osteoblast differentiation and block osteoclast activation, and to rebalance bone formation and resorption. Preclinical investigations on MSC transplantation in the osteoporosis treatment provide evidences of enhancing osteogenic differentiation, increasing bone mineral density, and halting the deterioration of osteoporosis. Meanwhile, the latest techniques, such as gene modification, targeted modification and co‐transplantation, are promising approaches to enhance the therapeutic effect and efficacy of MSCs. In addition, clinical trials of MSC therapy to treat osteoporosis are underway, which will fill the gap of clinical data. Although MSCs tend to be effective to treat osteoporosis, the urgent issues of safety, transplant efficiency and standardization of the manufacturing process have to be settled. Moreover, a comprehensive evaluation of clinical trials, including safety and efficacy, is still needed as an important basis for clinical translation.     

Exosomes derived from human umbilical cord mesenchymal stem cells alleviate acute liver failure by reducing the activity of the NLRP3 inflammasome in macrophages

Human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-EXOs) play an important role in the regulation of the immune system and inflammatory responses; however, their role in acute liver failure (ALF) and related pathological conditions is unclear. In this study, we found that hUCMSC-EXOs can reduce the expression of the NLRP3 inflammasome and downstream inflammatory factors in acute liver failure. Western blot and ELISA results showed that hUCMSC-EXOs decreased the expression of NLRP3, caspase-1, IL-1β and IL-6 in LPS-stimulated RAW 264.7 macrophages. In vivo, the hUCMSC-EXOs repaired damaged liver tissue and decreased the expression of the NLRP3 inflammasome and the levels of ALT and AST in a mouse ALF model. The results of this study provide a new strategy for the application of human umbilical cord mesenchymal stem cell-derived exosomes in the treatment of ALF.