Mesenchymal stem cells: Molecular characteristics and clinical applications

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells with the capacity to differentiate into tissues of both mesenchymal and non-mesenchymal origin. MSCs can differentiate into osteoblastic, chondrogenic, and adipogenic lineages, although recent studies have demonstrated that MSCs are also able to differentiate into other lineages, including neuronal and cardiomyogenic lineages. Since their original isolation from the bone marrow, MSCs have been successfully harvested from many other tissues. Their ease of isolation and ex vivo expansion combined with their immunoprivileged nature has made these cells popular candidates for stem cell therapies. These cells have the potential to alter disease pathophysiology through many modalities including cytokine secretion, capacity to differentiate along various lineages, immune modulation and direct cell-cell interaction with diseased tissue. Here we first review basic features of MSC biology including MSC characteristics in culture, homing mechanisms, differentiation capabilities and immune modulation. We then highlight some in vivo and clinical evidence supporting the therapeutic roles of MSCs and their uses in orthopedic, autoimmune, and ischemic disorders.     

Wnt signaling and skeletal development

Wnt proteins are a family of secreted proteins that regulate many aspects of cellular functions. The discovery that mutations in low-density lipoprotein receptor-related protein 5, a putative Wnt coreceptor, could positively and negatively affect bone mass in humans generated an enormous amount of interest in the possible role of the Wnt signaling pathway in skeletal biology. Over the last decade, considerable progress has been made in determining the role of the canonical Wnt signaling pathway in various aspects of skeletal development. Furthermore, recent evidence indicates the important role of non-canonical Wnt signaling in skeletal development. In this review we discuss the current understanding of the role of Wnt signaling in chondrogenesis, osteoblastogenesis, and osteoclastogenesis.     

Wnt通路中蓬松蛋白DNA甲基化对骨质疏松患者骨髓间充质干细胞成骨分化影响

摘要 目的：探讨蓬松蛋白（DVL）DNA甲基化对骨质疏松（OP）患者骨髓间充质干细胞（BMSCs）成骨分化及Wnt通路的影响。方法：分离、培养OP患者的BMSCs,成骨诱导培养BMSCs 0、7、14、21天,观察BMSCs细胞形态变化,检测碱性磷酸酶（ALP）染色及活性,检测茜素红染色及钙结节形成,荧光定量聚合酶链式反应（RT-PCR）及免疫印迹检测远端缺失同源盒5（Dlx5）、核心结合蛋白因子2（Runx2）、成骨细胞特异性转录因子（OSX）、Ⅰ型胶原蛋白（Colla Ⅰ）表达。检测DVL1、Wnt、糖原合成酶激酶3（GSK3）、β连环蛋白（β-catenin）表达及DVL DNA甲基化水平。在成骨诱导培养基中加入甲基转移酶抑制剂5-Aza,将BMSCs分为对照组（Control组）、甲基转移酶抑制剂组（5-Aza组）、甲基转移酶抑制剂+si-NC组（5-Aza+si-NC组）、甲基转移酶抑制剂+si-Wnt组（5-Aza+si-Wnt组）,依次进行ALP活性测定,茜素红染色及钙结节形成测定。RT-PCR检测Dlx5、Runx2、OSX、Colla 1水平,免疫印迹检测Dlx5、Runx2、OSX、Colla Ⅰ、DVL1、Wnt、GSK3、β-catenin的蛋白表达量,并检测DVL DNA甲基化水平。结果：成骨诱导后BMSCs具有强的ALP活性和矿化结节生成能力,且随着培养时间的增长,BMSCs细胞ALP活性和矿化结节生成能力增强,Dlx5、Runx2、OSX、Colla Ⅰ mRNA水平和Wnt、GSK3、β-catenin、DVL1表达升高,DVL DNA甲基化水平降低（P＜0.05）。5-Aza组较Control组ALP染色加深,活性增强,钙结节形成增多（P＜0.05）,Dlx5、Runx2、OSX、Colla Ⅰ mRNA及蛋白表达、Wnt、GSK3、β-catenin、DVL1表达升高,DVL DNA甲基化水平降低（P＜0.05）。5-Aza+si-Wnt组较5-Aza+si-NC组ALP染色变浅,活性降低,钙结节形成减少（P＜0.05）,Dlx5、Runx2、OSX、Colla Ⅰ mRNA及蛋白表达、Wnt、GSK3、β-catenin、DVL1表达降低,DVL DNA甲基化水平升高（P＜0.05）。结论：DVL DNA甲基化可以通过抑制Wnt/β-catenin信号通路抑制OP患者BMSCs成骨分化。     

Wnt 信号通路与骨髓间充质干细胞成骨之间的关系

Wnt信号通路是由Wnts诱发的一系列相互作用的分子组成。Wnt信号对骨髓间充质干细胞的影响在所有研究中均证实有明显作用,其可调节干细胞增殖、分化及凋亡。研究表明,抑制Wnt信号通路转导可使成骨细胞分化进程受阻,从而抑制骨形成;若诱导Wnt家族成员表达则可使成骨细胞特异性基因表达增加,促进骨形成。本文就Wnt信号通路的作用过程及其与骨髓间充质干细胞成骨诱导的关系做一综述。     

Non-hematopoietic bone marrow stem cells: molecular control of expansion and differentiation

The first non-hematopoietic mesenchymal stem cells (MSCs) were discovered by Friedenstein in 1976, who described clonal, plastic adherent cells from bone marrow capable of differentiating into osteoblasts, adipocytes, and chondrocytes. More recently, investigators have now demonstrated that multi-potent MSCs can be recovered from a variety of other adult tissues and differentiate into numerous tissue lineages including myoblasts, hepatocytes and possibly even neural tissue. Because MSCs are multipotent and easily expanded in culture, there has been much interest in their clinical potential for tissue repair and gene therapy and as a result, numerous studies have been carried out demonstrating the migration and multi-organ engraftment potential of MSCs in animal models and in human clinical trials. This review describes the recent advances in the understanding of MSC biology.     

Differentiating multipotent mesenchymal stromal cells generate factors that exert paracrine activities on exogenous MSCs: Implications for paracrine activities in bone regeneration

The mechanisms by which multipotent mesenchymal stromal cells (MSCs) contribute to tissue repair following transplantation into host tissues remains poorly understood. Current concepts suggest that, in addition to differentiation into cells of the host tissues, MSCs also generate trophic factors that modulate host tissue microenvironment to aid in the repair process. In this communication, we assessed whether factors secreted by MSCs undergoing osteogenic differentiation induce expression of osteoblast markers in exogenous MSCs as well as their migration. Murine MSCs were cultured in osteogenic medium, and at different time points, medium conditioned by the cells was collected and assessed for its effects on differentiation and migration of exogenous MSCs. In addition, we determined whether MSCs infused into mice femurs expressed genes encoding for factors predicted to play a role in paracrine activities. The results showed that MSCs maintained in osteogenic medium, secreted factors at specific time points that induced alkaline phosphatase activity (ALP) in exogenous MSCs as well as their migration. MSCs infused into mice femurs and retrieved at different days expressed genes that encoded predicted factors that play a role in cell differentiation and migration. Neutralizing antibodies to bone morphogenetic protein-2 (BMP-2) led to the decrease in ALP activity by exogenous MSCs. These data demonstrated that, as MSCs differentiate toward osteogenic lineage, they secrete factors that induce recruitment and differentiation of endogenous progenitors. These data reveal mechanisms by which donor MSCs may contribute to the bone reparative process and provide a platform for designing approaches for stem cell therapies of musculoskeletal disorders.     

芪丹通脉片促进骨髓间充质干细胞向缺血心肌趋化迁移

目的:研究芪丹通脉片对骨髓间充质干细胞向缺血心肌趋化迁移的作用。方法:SD大鼠,随机分为单纯移植组,益气组,活血组。芪丹通脉片组,各组大鼠灌服中药14天后,采用冠状动脉左前降支结扎法建立心肌梗死模型。经尾静脉注入DIO标记的骨髓间充质干细胞,3天后取缺血部位的心肌,应用流式细胞仪分析计算出每克心肌所含有的DIO阳性细胞数;行冰冻切片,采用荧光显微镜观察DIO阳性细胞;4周后采用多导生理记录仪记录大鼠平均动脉压(MAP)、左心室收缩峰压(LVPSP)、左心室内压最大上升变化速率(+LVdp/dtmax)和最大下降速率(-LVdp/dtmax)。结果:芪丹通脉片组较益气组、活血组、单纯移植组,每克心肌所含有的DIO阳性细胞数增多,其差异有统计学意义;冰冻切片镜下观察,芪丹通脉片组阳性细胞数较益气组、活血组、单纯移植组增多,其差异有统计学意义;心功能检测示:芪丹通脉片组MAP、LVPSP、+LVdp/dtmax、-LVdp/dtmax较益气组、活血组、单纯移植组改善明显,差异有统计学意义。结论:芪丹通脉片具有促进骨髓间充质干细胞向缺血心肌趋化迁移的作用,益气与活血中药配伍应用优于单纯益气药或活血药。     

干细胞改善糖尿病的分子机制及临床研究进展

糖尿病是各种因素导致的高血糖慢性代谢疾病,已发展成为流行疾病之一。化学抗糖药虽能控制血糖水平,延缓病程进展,但需长期服用;胰岛移植能从根本上治愈糖尿病,但胰岛来源不足,且需终生应用免疫抑制剂,故并没有得到广泛应用;干细胞是一类能够自我复制的细胞,具有多向分化潜能和旁分泌特性,近年来的研究证明,干细胞在糖尿病治疗方面有着积极的效果,被认为是有效治疗糖尿病的理想细胞类型。因此,就干细胞治疗糖尿病的分子机制和临床研究现状进行简要阐述。     

小型猪骨髓间充质干细胞的分离和培养

目的建立小型猪骨髓间充质干细胞（mesenchymal stem cells,MSCs）的体外分离和培养方法。方法穿刺小型猪髂后上嵴抽取骨髓,经密度梯度法离心得到骨髓单个核细胞,接种后形成单层贴壁细胞。用形态学方法鉴定培养的MSCs。结果经培养存活的MSCs原代和一代呈纺锤型、多边型或星型。至二代起呈均一纺锤型,似成纤维细胞样,长宽比例约为（2～3）？1。体外培养的原代MSCs8～10d达到融合,传代后仍具有较强的增殖能力。结论小型猪MSCs可在体外长期、稳定培养,其分离、培养体系的建立为基础研究和组织工程技术提供了一个有价值的动物模型。     

Current evidence on potential of adipose derived stem cells to enhance bone regeneration and future projection

Injuries to the postnatal skeleton are naturally repaired through successive steps involving specific cell types in a process collectively termed “bone regeneration”. Although complex, bone regeneration occurs through a series of well-orchestrated stages wherein endogenous bone stem cells play a central role. In most situations, bone regeneration is successful; however, there are instances when it fails and creates non-healing injuries or fracture nonunion requiring surgical or therapeutic interventions. Transplantation of adult or mesenchymal stem cells (MSCs) defined by the International Society for Cell and Gene Therapy (ISCT) as CD105+CD90+CD73+CD45-CD34-CD14orCD11b-CD79αorCD19-HLA-DR- is being investigated as an attractive therapy for bone regeneration throughout the world. MSCs isolated from adipose tissue, adipose-derived stem cells (ADSCs), are gaining increasing attention since this is the most abundant source of adult stem cells and the isolation process for ADSCs is straightforward. Currently, there is not a single Food and Drug Administration (FDA) approved ADSCs product for bone regeneration. Although the safety of ADSCs is established from their usage in numerous clinical trials, the bone-forming potential of ADSCs and MSCs, in general, is highly controversial. Growing evidence suggests that the ISCT defined phenotype may not represent bona fide osteoprogenitors. Transplantation of both ADSCs and the CD105^- sub-population of ADSCs has been reported to induce bone regeneration. Most notably, cells expressing other markers such as CD146, AlphaV, CD200, PDPN, CD164, CXCR4, and PDGFRα have been shown to represent osteogenic sub-population within ADSCs. Amongst other strategies to improve the bone-forming ability of ADSCs, modulation of VEGF, TGF-β1 and BMP signaling pathways of ADSCs has shown promising results. The U.S. FDA reveals that 73% of Investigational New Drug applications for stem cell-based products rely on CD105 expression as the “positive” marker for adult stem cells. A concerted effort involving the scientific community, clinicians, industries, and regulatory bodies to redefine ADSCs using powerful selection markers and strategies to modulate signaling pathways of ADSCs will speed up the therapeutic use of ADSCs for bone regeneration.     

Stem cell-derived exosomes as a therapeutic tool for cardiovascular disease

Mesenchymal stem cells(MSCs) have been used to treat patients suffering from acute myocardial infarction(AMI) and subsequent heart failure. Although it was originally assumed that MSCs differentiated into heart cells such as cardiomyocytes, recent evidence suggests that the differentiation capacity of MSCs is minimal and that injected MSCs restore cardiac function via the secretion of paracrine factors. MSCs secrete paracrine factors in not only naked forms but also membrane vesicles including exosomes containing bioactive substances such as proteins, messenger RNAs, and microR NAs. Although the details remain unclear, these bioactive molecules are selectively sorted in exosomes that are then released from donor cells in a regulated manner. Furthermore, exosomes are specifically internalized by recipient cells via ligand-receptor interactions. Thus, exosomes are promising natural vehicles that stably and specifically transport bioactive molecules to recipient cells. Indeed, stem cell-derived exosomes have been successfully used to treat cardiovascular disease(CVD), such as AMI, stroke, and pulmonary hypertension, in animal models, and their efficacy has been demonstrated. Therefore, exosome administration may be a promising strategy for the treatment of CVD. Furthermore, modifications of exosomal contents may enhance their therapeutic effects. Future clinical studies are required to confirm the efficacy of exosome treatment for CVD.     

Notch信号参与BMP4诱导的间充质干细胞成骨分化及其机制的初步探讨

目的:探讨Notch信号对骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)诱导间充质干细胞成骨分化的影响以及作用机制。方法:(1)DAPT或Ad-dominant-negative mutants of Notch1(Addn Notch1)和BMP4-CM处理小鼠胚胎成纤维细胞,检测早期成骨指标碱性磷酸酶(alkaline phosphatase,ALP);(2)茜素红S染色实验检测晚期成骨钙盐沉积情况;(3)半定量反转录聚合酶链反应(RT-PCR)检测成骨分化相关基因ALP,Runx2,Col1a1的表达;(4)免疫细胞化学检测p-Smad1/5/8的表达;(5)结晶紫染色和流式细胞术检测细胞的增殖及周期改变。结果:(1)DAPT抑制BMP4诱导的早期成骨分化,且呈浓度依赖性;(2)Delta-like 1(DLL1)促进BMP4诱导的成骨分化,DAPT和dn Notch1抑制BMP4诱导的成骨分化;(3)DLL1促进BMP4诱导的成骨相关基因ALP,Runx2,Col1a1的表达,DAPT抑制这些基因的表达;(4)DLL1促进BMP4诱导的细胞核内p-Smad1/5/8的表达,而DAPT抑制其表达;(5)DLL1促进BMP4诱导的细胞增殖,而DAPT抑制BMP4诱导的细胞增殖。结论:Notch信号通过BMP/Smads信号通路促进BMP4诱导的MSCs成骨分化,在此过程中也有促细胞增殖的作用。     

Mesenchymal stem cell-derived exosomes: Toward cell-free therapeutic strategies in regenerative medicine

Mesenchymal stem cells (MSCs) are multipotent stem cells with marked potential for regenerative medicine because of their strong immunosuppressive and regenerative abilities. The therapeutic effects of MSCs are based in part on their secretion of biologically active factors in extracellular vesicles known as exosomes. Exosomes have a diameter of 30-100 nm and mediate intercellular communication and material exchange. MSC-derived exosomes (MSC-Exos) have potential for cell-free therapy for diseases of, for instance, the kidney, liver, heart, nervous system, and musculoskeletal system. Hence, MSC-Exos are an alternative to MSC-based therapy for regenerative medicine. We review MSC-Exos and their therapeutic potential for a variety of diseases and injuries.     

脐静脉和骨髓来源的间充质干细胞的比较研究

间充质干细胞(MSCs)的来源有限,成人骨髓是MSCs的主要来源,这极大地限制了其在实验和临床中的应用。为拓宽MSCs来源,从细胞形态、生长特性、免疫表型和多向分化能力等四个方面对人脐静脉来源和成人骨髓来源的间充质干细胞进行了比较研究。结果表明,人脐静脉来源和成人骨髓来源的 MSCs具有相似的生物学特征,成纤维细胞样形态生长,并具有强大的体外扩增和多向分化能力。人脐静脉来源的MSCs可替代成人骨髓MSCs,作为满足实验和临床需要的重要来源。     

Mesenchymal stem cells originating from ES cells show high telomerase activity and therapeutic benefits

We establish a novel method for the induction and collection of mesenchymal stem cells using a typical cell surface marker, CD105, through adipogenesis from mouse ES cells. ES cells were cultured in a medium for adipogenesis. Mesenchymal stem cells from mouse ES cells were easily identified by the expression of CD105, and were isolated and differentiated into multiple mesenchymal cell types. Mesenchymal stem cells showed remarkable telomerase activity and sustained their growth for a long time with a high potential for differentiation involving skeletal myogenesis in vitro. When mesenchymal stem cells were transplanted into the injured tibialis anterior muscles, they differentiated into skeletal muscle cells in vivo. In addition, they improved the vascular formation, but never formed teratoma for longer than 6 months. Gene expression profiles revealed that mesenchymal stem cells lost pluripotency, while they acquired high potential to differentiate into mesenchymal cell lines. They thus indicate a promising new source of cell-based therapy without teratoma formation.     

骨髓间充质干细胞复合支架材料的骨组织工程研究进展

近年来,组织工程技术飞速发展,将种子细胞与支架材料相复合的骨组织工程研究已成为热点,并日趋走向成熟。这一全新的治疗方案将成为解决临床上各种原因造成的骨组织缺损的最有效途径之一。骨组织工程技术包括种子细胞、支架材料和生长因子三个方面。其中,BMSCs因具有多向分化能力、强大的增殖能力以及低免疫源性被认为是最理想的种子细胞,而支架材料的种类有很多种,目前对支架材料的选择也尚有分歧。如何找到理想的支架材料是骨组织工程研究中亟待解决的重要问题。本文就组织工程中与骨髓间充质干细胞(BMSCs)相复合的各类支架材料的研究现状进行综述,这些支架材料的研究将为骨组织工程支架材料的选择提供有效依据。     

小鼠骨髓间充质干细胞的分离、培养及鉴定

目的建立一种从小鼠骨髓中分离培养间充质干细胞（MSCs）的高效方法。方法采取贴壁细胞分离法分离和纯化小鼠骨髓间充质干细胞（mMSCs）,检测mMSCs在不同诱导条件下向成骨细胞及脂肪细胞分化能力,用流式细胞术及显微镜分别检测mMSCs纯度和形态特征。结果mMSCs贴壁生长后形态较均一,细胞形态呈成纤维细胞样,流式细胞术检测：CD45、CD11b、CD44及CD29分别为（3.34）%、（2.41）%、（98.46）%及（99.36）%。第4代mMSCs经诱导后可向成骨细胞和脂肪细胞分化。结论通过贴壁培养可以从小鼠骨髓中分离培养出高纯度mMSCs,该方法效率高,稳定性好。