Induction of factor VIII‐specific unresponsiveness by intrathymic factor VIII injection in murine hemophilia A

Summary. Background: Hemophilia A is a congenital bleeding disorder caused by a deficiency of coagulation factor VIII. Approximately 30% of hemophilia A patients develop inhibitors against FVIII following replacement therapy. We have reported that neonatal exposure of FVIII antigen can induce antigen‐specific immune tolerance by interferon‐γ (IFN‐γ)‐dependent T‐cell anergy in hemophilia A mice. Objective: The thymus plays crucial roles in self‐tolerance, with negative selection of self‐reactive effector T cells and positive selection of self‐reactive regulatory T cells. We investigated the possibility of the induction of antigen‐specific immune tolerance by intrathymic injection of FVIII in hemophilia A mice. Methods: Hemophilia A mice were injected with recombinant FVIII into the thymus under real‐time high‐resolution image guidance. Results: Anti‐FVIII inhibitory antibody titers in mice challenged with intravenous administration of FVIII were significantly lower in mice (n = 22) that had received thymic FVIII injection than in mice (n = 18) without thymic injection (9.4 ± 2.3 vs. 122.5 ± 27.6 BU mL^?1, respectively, P = 0.00078). The CD4⁺ T cells from thymic‐injected mice could not proliferate or produce interleukin (IL)‐2, IL‐12 and IFN‐γ in response to FVIII. The CD4⁺CD25⁺ T cells generated from thymic‐treated mice but not from naïve mice efficiently suppressed the in vitro proliferative response of CD4⁺ T cells and blocked the in vivo development of anti‐FVIII antibodies in the adoptive transfer. Conclusion: These data suggest that intrathymic administration of FVIII could result in immune tolerance by induction of FVIII‐specific regulatory T cells.     

基因修饰骨髓间充质干细胞修复关节软骨损伤

背景：随着生物技术的发展,通过转基因技术修饰细胞,从而获得长期稳定表达的生物活性因子以治疗关节软骨损伤逐渐引起重视。目的：就基因修饰的骨髓间充质干细胞在修复关节软骨损伤中的应用作一综述。方法：由第一作者检索1990至2011年PubMed数据库（http：／／www．ncbi．nlm．nih.gov／PubMed）有关基因修饰骨髓间充质干细胞修复关节软骨损伤的文献,英文检索词为“cartilage,genetherapy,mesenchymalstemcells,tissueengineering,bioactivefactor,vector”。共纳入15篇文献归纳总结。结果与结论：骨髓间充质干细胞已被广泛应用于修复关节软骨损伤。通过转基因技术将特定外源基因导入骨髓间充质干细胞,联合细胞治疗和基因治疗可达到更好的治疗效果,在关节软骨损伤的治疗中有广阔的应用前景。     

Enhanced treatment of articular cartilage defect of the knee by intra‐articular injection of Bcl‐xL‐engineered mesenchymal stem cells in rabbit model

Direct intra‐articular injection of mesenchymal stem cells (MSCs) has been proposed as a potential cell therapy for cartilage defects. This cell therapy relies on the survival of the implanted MSCs. However, the arduous local environment may limit cell viability after implantation, which would restrict the cells' regenerative capacity. Thus, it is necessary to reinforce the implanted cells against the unfavourable microenvironment in order to improve the efficacy of cell therapy. We examined whether the transduction of an anti‐apoptotic protein, Bcl‐xL, into MSCs could prevent cell death and improve the implantation efficiency of MSCs in a rabbit model. Our current findings demonstrate that the group treated with Bcl‐xL‐engineered MSCs could improve cartilage healing both morphologically and histologically when compared with the controls. These results suggest that intra‐articular injection of Bcl‐xL‐engineered MSCs is a potential non‐invasive therapeutic method for effectively treating cartilage defects of the knee. Copyright © 2009 John Wiley & Sons, Ltd.     

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases

The intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells, including the pluripotent haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and possibly the multipotent adherent progenitor cells (MAPCs), are being considered as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and, at least in vitro, have significant expansion capability. The apparently high self-renewal potential makes them strong candidates for delivering genes and restoring organ systems function. However, the high proliferative potential of MSCs, now presumed to be self-renewal, may be more apparent than real. Although expanded MSCs have great proliferation and differentiation potential in vitro, there are limitations with the biology of these cells in vivo. So far, expanded MSCs have failed to induce durable therapeutic effects expected from a true self-renewing stem cell population. The loss of in vivo self-renewal may be due to the extensive expansion of MSCs in existing in vitro expansion systems, suggesting that the original stem cell population and/or properties may no longer exist. Rather, the expanded population may indeed be heterogeneous and represents several generations of different types of mesenchymal cell progeny that have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and non-mesenchymal lineages. Novel technology that allows MSCs to maintain their stem cell function in vivo is critical for distinguishing the elusive stem cell from its progenitor cell populations. The ultimate dream is to use MSCs in various forms of cellular therapies, as well as genetic tools that can be used to better understand the mechanisms leading to repair and regeneration of damaged or diseased tissues and organs.     

骨髓间充质干细胞诱导成骨细胞模型中活化转录因子4基因的表达

背景：研究发现活化转录因子4为近调控成骨细胞分化和功能的活化因子,在成骨细胞分化过程中起着至关重要的作用。 目的：探索SD大鼠骨髓间充质干细胞成骨诱导过程中活化转录因子4基因的表达变化及其意义。 方法：以全骨髓贴壁法培养SD大鼠骨髓间充质干细胞,传代至第3代细胞时加入成骨诱导剂,在诱导的第0,1,4,7,10,13,16,19天分别采用PT-PCR,Western blot动态监测活化转录因子4基因及蛋白的表达变化,以未进行成骨诱导细胞做对照。 结果与结论：RT-PCR结果显示,活化转录因子4 mRNA的表达随着细胞成骨分化程度加剧而升高,16 d达到峰值。Western blot分析结果显示,活化转录因子4蛋白表达量随着骨髓间充质干细胞骨化程度加剧,表达水平亦呈上升趋势,于16 d达到高峰,19 d维持在高水平状态。实验组各时间点活化转录因子4 mRNA和活化转录因子4蛋白表达与对照组相比差异有均有显著性意义（P＆lt;0.05）。结果表明诱导成骨细胞中活化转录因子4表达增强,提示活化转录因子4的增强与骨髓间充质干细胞成骨分化能力呈正相关。     

Differential bone‐forming capacity of osteogenic cells from either embryonic stem cells or bone marrow‐derived mesenchymal stem cells

For more than a decade, human mesenchymal stem cells (hMSCs) have been used in bone tissue‐engineering research. More recently some of the focus in this field has shifted towards the use of embryonic stem cells. While it is well known that hMSCs are able to form bone when implanted subcutaneously in immune‐deficient mice, the osteogenic potential of embryonic stem cells has been mainly assessed in vitro. Therefore, we performed a series of studies to compare the in vitro and in vivo osteogenic capacities of human and mouse embryonic stem cells to those of hMSCs. Embryonic and mesenchymal stem cells showed all characteristic signs of osteogenic differentiation in vitro when cultured in osteogenic medium, including the deposition of a mineralized matrix and expression of genes involved in osteogenic differentiation. As such, based on the in vitro results, osteogenic ES cells could not be discriminated from osteogenic hMSCs. Nevertheless, although osteogenic hMSCs formed bone upon implantation, osteogenic cells derived from both human and mouse embryonic stem cells did not form functional bone, indicated by absence of osteocytes, bone marrow and lamellar bone. Although embryonic stem cells show all signs of osteogenic differentiation in vitro, it appears that, in contrast to mesenchymal stem cells, they do not possess the ability to form bone in vivo when a similar culture method and osteogenic differentiation protocol was applied. Copyright © 2010 John Wiley & Sons, Ltd.     

Persistence of fluorescent nanoparticle‐labelled bone marrow mesenchymal stem cells in vitro and after intra‐articular injection

Mesenchymal stem cells (MSCs) improve the osteoarthritis condition, but the fate of MSCs after intra‐articular injection is unclear. We used fluorescent nanoparticles (quantum dots [QDs]) to track equine MSCs (QD‐labelled MSCs [QD‐MSCs]) in vivo after intra‐articular injection into normal and osteoarthritic joints. One week after injection of QD‐MSCs, unlabelled MSCs, or vehicle, we determined the presence of QD‐MSCs in synovium and articular cartilage histologically. In vitro, we evaluated the persistence of QDs in MSCs and whether QDs affected proliferation, immunophenotype, or differentiation. In joints injected with QD‐MSCs, labelled cells were identified on the synovial membrane and significantly less often on articular cartilage, without differences between normal and osteoarthritic joints. Joints injected with QD‐MSCs and MSCs had increased synovial total nucleated cell count and protein compared with vehicle‐injected joints. In vitro, QDs persisted in nonproliferating cells for up to 8 weeks (length of the study), but QD fluorescence was essentially absent from proliferating cells within two passages (approximately 3 to 5 days). QD labelling did not affect MSC differentiation into chondrocytes, adipocytes, and osteocytes. QD‐MSCs had slightly different immunophenotype from control cells, but whether this was due to an effect of the QDs or to drift during culture is unknown. QD‐MSCs can be visualized in histological sections 1 week after intra‐articular injection and are more frequently found in the synovial membrane versus cartilage in both normal and osteoarthritic joints. QDs do not alter MSC viability and differentiation potential in vitro. However, QDs are not optimal markers for long‐term tracking of MSCs, especially under proliferative conditions.     

Matrilin‐3 codelivery with adipose‐derived mesenchymal stem cells promotes articular cartilage regeneration in a rat osteochondral defect model

Matrilin‐3 is an essential extracellular matrix component present only in cartilaginous tissues. Matrilin‐3 exerts chondroprotective effects by regulating an anti‐inflammatory function and extracellular matrix components. We hypothesized that the codelivery of matrilin‐3 with infrapatellar adipose‐tissue‐derived mesenchymal stem cells (Ad‐MSCs) may enhance articular cartilage regeneration. Matrilin‐3 treatment of Ad‐MSCs in serum‐free media induced collagen II and aggrecan expression, and matrilin‐3 in chondrogenic media also enhanced in vitro chondrogenic differentiation. Next, the in vivo effect of matrilin‐3 codelivery with Ad‐MSCs on cartilage regeneration was assessed in an osteochondral defect model in Sprague Dawley rats: Ad‐MSCs and hyaluronic acid were implanted at the defect site with or without matrilin‐3 (140, 280, and 700 ng). Safranin O staining revealed that matrilin‐3 (140 and 280 ng) treatment significantly improved cartilage regeneration and glycosaminoglycan accumulation. In the animals treated with 140‐ng matrilin‐3, in particular, the defect site exhibited complete integration with surrounding tissue and a smooth glistening surface. The International Cartilage Repair Society macroscopic and O'Driscoll microscopic scores for regenerated cartilage were furthermore shown to be considerably higher for this group (matrilin‐3; 140 ng) compared with the other groups. Furthermore, the defects treated with 140‐ng matrilin‐3 revealed significant hyaline‐like cartilage regeneration in the osteochondral defect model; in contrast, the defects treated with 700‐ng matrilin‐3 exhibited drastically reduced cartilage regeneration with mixed hyaline–fibrocartilage morphology. Codelivery of matrilin‐3 with Ad‐MSCs significantly influenced articular cartilage regeneration, supporting the potential use of this tissue‐specific protein for a cartilage‐targeted stem cell therapy.     

组蛋白去乙酰化酶在卵巢切除小鼠骨髓间充质干细胞中的表达

背景：骨髓间充质干细胞的多向分化能力在骨代谢疾病中发挥重要作用,受激素、细胞因子等多种因素调节。目前骨髓间充质干细胞骨向分化的表观遗传学调控机制尚不明确,组蛋白去乙酰化酶与骨质疏松的关系尚需进一步探讨。 目的：建立雌激素缺乏骨质疏松小鼠的实验动物模型,检测骨髓间充质干细胞组蛋白去乙酰化酶1,3,4 mRNA表达水平,探索卵巢切除小鼠骨组织形成障碍的表观遗传学机制。 方法：昆明种小鼠30只随机等分为模型组和假手术组。小鼠适应性喂养7 d后,模型组小鼠切除双侧卵巢,造成雌激素缺乏骨质疏松实验动物模型,假手术组小鼠仅切除等量脂肪组织。 结果与结论：模型组小鼠股骨骨小梁稀疏或断裂,骨小梁宽度变窄,骨小梁间距变宽,骨小梁占视野面积降低。与假手术组相比,模型组小鼠骨髓间充质干细胞中组蛋白去乙酰化酶3 mRNA表达水平显著降低,组蛋白去乙酰化酶1,4表达水平变化差异无显著性意义。提示雌激素缺乏导致骨髓间充质干细胞去乙酰化状态改变可能是骨形成障碍的重要原因之一。     

间充质干细胞在关节软骨组织工程中的应用

背景：组织工程技术的发展为关节软骨缺损修复和功能重建提供了新的方法和思路。目的：探讨以间充质干细胞作为种子细胞在关节软骨组织工程中的应用和研究进展。 方法：由第一作者检索 PubMed 数据库中2000-01-01/2014-09-30有关间充质干细胞和关节软骨组织工程的文献,检索词为“articular cartilage defects, cartilage tissue engineering, mesenchymal stem cel s”。共检索到70篇相关文献,对其中49篇文献进行综述。 结果与结论：关节软骨缺损自身修复能力很有限,目前的临床治疗手段无法达到满意修复,而组织工程的发展为解决这个问题提供了新思路。在种子细胞选择方面,软骨细胞去分化能力有限,胚胎干细胞受到伦理、法律等方面的制约,而间充质干细胞因其自体来源、易扩增、具有软骨分化潜能而受到广泛重视。但目前应用组织工程方法修复关节软骨缺损的效果存在一定的争议,主要是远期功能距离临床应用存在一定差距,在修复组织结构和生物力学方面还需要进一步研究。     

Chondrogenic potential of human mesenchymal stem cells and expression of Slug transcription factor

The scientific literature rarely reports experimental failures or inconsistent outcomes in the induction of cell differentiation; however, researchers commonly experience poor or unsuccessful responses to differentiating agents when culturing stem cells. One way of investigating the underlying reasons for such responses is to look at the basal expression levels of specific genes in multipotent stem cells before the induction of differentiation. In addition to shedding light on the complex properties of stem cells and the molecular modulation of differentiation pathways, this strategy can also lead to the development of important time‐ and money‐saving tools that aid the efficient selection of cellular specimens – in this case, stem cells that are more prone to differentiate towards specific lineages and are therefore more suitable for cell‐based therapeutic protocols in regenerative medicine. To address this latter aspect, this study focused on understanding the reasons why some human mesenchymal stem cell (hMSC) samples are less efficient at differentiating towards chondrogenesis. This study shows that analysis of the basal expression levels of Slug, a negative regulator of chondrogenesis in hMSC, provides a rapid and simple tool for distinguishing stem cell samples with the potential to form a cartilage‐like matrix, and that are therefore suitable for cartilage tissue engineering. It is shown that high basal levels of Slug prevent the chondrogenic differentiation of hMSCs, even in the presence of transforming growth factor‐β and elevated levels of Sox9. Copyright © 2013 John Wiley & Sons, Ltd.     

Donor‐matched comparison of dental pulp stem cells and bone marrow‐derived mesenchymal stem cells in a rat model

Dental pulp stem cells (DPSCs) have drawn much interest for the regeneration of mineralized tissues, and several studies have compared DPSCs to bone marrow‐derived mesenchymal stem cells (BMMSCs). However, conflicting results, possibly due to donor‐associated variability, have been published and the regenerative potential of DPSCs is currently unclear. In the present study we have sought to address this problem using a donor‐matched experimental design to robustly compare the biological properties of DPSCs and BMMSCs. All experiments were performed using cells isolated from a single adult Sprague–Dawley rat. Our results show that DPSCs and BMMSCs had similar morphologies and flow cytometry profiles, were capable of forming colonies in vitro and were capable of osteogenic, chondrogenic and adipogenic differentiation. However, quantitative comparisons revealed that DPSCs had a faster population doubling time and a higher percentage of stem/progenitor cells in the population, as determined by clonogenic assays. Furthermore, while both cell populations formed mineral in vitro, DPSCs had significantly higher alkaline phosphatase activity than BMMSCs after 3 weeks in osteogenic medium. These data show several key differences between DPSCs and BMMSCs and support the possibility of using DPSCs for mineralized tissue regeneration. Copyright © 2009 John Wiley & Sons, Ltd.     

Engineering vascularized flaps using adipose‐derived microvascular endothelial cells and mesenchymal stem cells

Human adipose‐derived microvascular endothelial cells (HAMEC) and mesenchymal stem cells (MSC) have been shown to bear angiogenic and vasculogenic capabilities. We hypothesize that co‐culturing HAMEC:MSC on a porous biodegradable scaffold in vitro, later implanted as a graft around femoral blood vessels in a rat, will result in its vascularization by host vessels, creating a functional vascular flap that can effectively treat a range of large full‐thickness soft tissue defects. HAMEC were co‐cultured with MSC on polymeric three‐dimensional porous constructs. Grafts were then implanted around the femoral vessels of a rat. To ensure vessel sprouting from the main femoral vessels, grafts were pre‐isolated from the surrounding tissue. Graft vascularization was monitored to confirm full vascularization before flap transfer. Flaps were then transferred to treat both abdominal wall and exposed bone and tendon of an ankle defects. Flaps were analysed to determine vascular properties in terms of maturity, functionality and survival of implanted cells. Findings show that pre‐isolated grafts bearing the HAMEC:MSC combination promoted formation of highly vascularized flaps, which were better integrated in both defect models. The results of this study show the essentiality of a specific adipose‐derived cell combination in successful graft vascularization and integration, two processes crucial for flap survival. Copyright © 2017 John Wiley & Sons, Ltd.     

MicroRNAs can effectively induce formation of insulin‐producing cells from mesenchymal stem cells

MicroRNAs regulate insulin secretion, pancreatic development and beta cell differentiation. However, the function of microRNAs in the formation of insulin‐producing cells (IPCs) from adult stem cells is poorly understood. We examine the microRNA expression profile in nestin‐positive umbilical cord‐derived mesenchymal stem cells (N‐UCMSCs) and nestin‐positive pancreatic mesenchymal stem cells using a deep sequencing approach. We also selected specific microRNAs for overexpression in N‐UCMSCs and found that miR‐375 and miR‐26a induced IPCs differentiation from N‐UCMSCs by downregulating target genes including mtpn, sox6, bhlhe22 and ccnd1. Small interfering RNAs were also used to knock down these genes in N‐UCMSCs to induce the formation of IPCs. These results suggest that endogenous microRNAs involved in the formation of IPCs from adult stem cells show promise for advancing the development of an effective cell transplant therapy for diabetes. Copyright © 2017 John Wiley & Sons, Ltd.     

LacZ转基因小鼠间充质干细胞的分离培养和生物学特性研究

摘要：目的：分离培养LacZ转基因小鼠骨髓间充质干细胞(LacZ-MSC),研究其生物学特性。 方法：用全骨髓贴壁法分离LacZ-MSC,体外扩增,并观察细胞生长特性,流式细胞仪检测细胞周期,成骨成脂诱导试验分析LacZ-MSC转分化特性, X-gal染色法检测其LacZ基因的表达。 结果：LacZ-MSC培养4 d后有散在呈针尖状的贴壁细胞,5~8 d后形成集落或融合呈纤维状;体外扩增每只小鼠原代可获得(1~3)×10⁵个细胞, 10代可获得(1~3)×10¹⁰个细胞总数,10代后的细胞增殖能力下降;细胞周期分析显示,G₀/G₁期：占78.44%,G₂/M期：占5.20%,S期：占16.36%;成骨成脂诱导试验表明,LacZ-MSC细胞具有向成骨细胞和脂肪细胞分化的潜能;X-gal染色结果表明,LacZ-MSC细胞携带LacZ基因。 结论:从LacZ转基因小鼠骨髓中成功地分离了携带有LacZ基因的MSC,在体外具有较好的增殖更新能力,3~10代的LacZMSC作为组织工程细胞具有广阔的应用前景。     

The neural plasticity of early‐passage human bone marrow‐derived mesenchymal stem cells and their modulation with chromatin‐modifying agents

Mesenchymal stem cells (MSCs) in their immature state express a variety of genes of the three germ layers at relatively low or moderate levels that might explain their phenomenal plasticity. Numerous recent studies have demonstrated that under the appropriate conditions in vitro and in vivo the expression of different sets of these genes can be upregulated, turning MSCs into variety of cell lineages of mesodermal, ectodermal and endodermal origin. While transdifferentiation of MSCs is still controversial, these unique properties make MSCs an ideal autologous source of easily reprogrammable cells. Recently, using the approach of cell reprogramming by biological active compounds that interfere with chromatin structure and function, as well as with specific signalling pathways that promote neural fate commitment, we have been able to generate neural‐like cells from human bone marrow (BM)‐derived MSCs (hMSCs). However, the efficiency of neural transformation of hMSCs induced by this approach gradually declined with passaging. To elucidate the mechanisms that underlie the higher plasticity of early‐passage hMSCs, comparative analysis of the expression levels of several pluripotent and neural genes was conducted for early‐ and late‐passage hMSCs. The results demonstrated that early‐passage hMSCs expressed the majority of these genes at low and moderate levels that gradually declined at late passages. Neural induction further increased the expression of some of these genes in hMSCs, accompanied by morphological changes into neural‐like cells. We concluded that low and moderate expression of several pluripotent and neural genes in early‐passage hMSCs could explain their higher plasticity and pliability for neural induction. Copyright © 2012 John Wiley & Sons, Ltd.