Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation

The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering.     

Allogeneic mesenchymal stem cells: agents of immune modulation

Adult mesenchymal stem cells possess a remarkably diverse array of immunosuppressive characteristics. The capacity to suppress the regular processes of allogeneic rejection, have allowed the use of tissue mismatched cells as therapeutic approaches in regenerative medicine and as agents of immune deviation. This review describes recent advances in understanding the mechanistic basis of mesenchymal stromal or stem cells (MSC) interaction with innate immunity. Particular emphasis is placed on the effect of Toll-like receptor signalling on MSC and a hypothesis that innate immune signals induce a 'licensing switch' in MSC is put forward. The mechanisms underlying MSC suppression of T cell responses and induction of regulatory populations are surveyed. Conflicting data regarding the influence of MSC on B cell function are outlined and discussed. Finally the limits to MSC mediated immune modulation are discussed with reference to the future clinical application of novel cell therapies.     

胚胎干细胞治疗心肌梗死的研究进展

胚胎干细胞 (ES细胞 )是一种多能细胞 ,来源于囊胚期胚胎 ,具有很强的自我更新能力 ,并能分化成很多细胞类型。体外 ,ES细胞能自发聚集形成胚胎体 (EB) ,分化成许多种细胞类型 ;ES细胞注射到免疫缺陷的小鼠体内 ,产生畸胎瘤 ,其中包含有三个胚层的细胞。添加生长因子或与其它细胞共培养等方法可以促进ES细胞体外分化为心肌细胞 ,筛选后移植到梗死的心肌 ,可以提高心脏功能 ,是治疗心肌梗死的一种很有潜力的方法     

Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells

The presence within bone marrow of a population of mesenchymal stem cells (MSCs) able to differentiate into a number of different mesenchymal tissues, including bone and cartilage, was first suggested by Friedenstein nearly 40 years ago. Since then MSCs have been demonstrated in a variety of fetal and adult tissues, including bone marrow, fetal blood and liver, cord blood, amniotic fluid and, in some circumstances, in adult peripheral blood. MSCs from all of these sources can be extensively expanded in vitro and when cultured under specific permissive conditions retain their ability to differentiate into multiple lineages including bone, cartilage, fat, muscle, nerve, glial and stromal cells. There has been great interest in these cells both because of their value as a model for studying the molecular basis of differentiation and because of their therapeutic potential for tissue repair and immune modulation. However, MSCs are a rare population in these tissues. Here we tried to identify cells with MSC-like potency in human placenta. We isolated adherent cells from trypsin-digested term placentas and examined these cells for morphology, surface markers, and differentiation potential and found that they expressed several stem cell markers. They also showed endothelial and neurogenic differentiation potentials under appropriate conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to MSCs in terms of morphology and cell-surface antigen expression. The placenta may prove to be a useful source of MSCs.     

Nano-particle engineered atorvastatin delivery to support mesenchymal stem cell survival in infarcted myocardium

Atorvastatin (ATV) may support mesenchymal stem cells (MSC) survival in post-infarct myocardium (MI) as inflammatory reactions, oxidative stress and hypoxia condition get started in such tissues after damage. However, limited aqueous insolubility and rapid first-pass metabolism reduce the systemic availability of ATV. The aim of the present investigation was to develop ATV loaded nanoparticles (ATVNPs) which might ensure the maximum availability of ATV in systemic circulation for longer duration and to strengthen the support to MSC survival. ATVNPs were synthesized using double emulsion solvent evaporation method and characterized as spherical shape, positive charged, nanoparticles of uniform size distribution and higher entrapment efficiency. ATVNPs were non-cytotoxic and showed sustained release (up to 28 days). Assessment of cardiac function (in terms of echocardiographic and left heart catheterization parameters) and cytokines estimation revealed efficient improvement in post-infarct myocardium condition of rat. In conclusion, ATVNP was developed successfully that may ensure safe, cost effective, and efficacious treatment of post-infarct myo-cardium when compared with that of MSC alone and MSC supplemented with ATV solution.     

Indocyanine green labeling for optical and photoacoustic imaging of mesenchymal stem cells after in vivo transplantation

The transplantation of mesenchymal stem cells (MSCs) holds great promise for the treatment of a plethora of human diseases, but new noninvasive procedures are needed to monitor the cell fate in vivo. Already largely used in medical diagnostics, the fluorescent dye indocyanine green (ICG) is an established dye to track limited numbers of cells by optical imaging (OI), but it can also be visualized by photoacoustic imaging (PAI), which provides a higher spatial resolution than pure near infrared fluorescence imaging (NIRF). Because of its successful use in clinical and preclinical examinations, we chose ICG as PAI cell labeling agent. Optimal incubation conditions were defined for an efficient and clinically translatable MSC labeling protocol, such that no cytotoxicity or alterations of the phenotypic profile were observed, and a consistent intracellular uptake of the molecule was achieved. Suspensions of ICG‐labeled cells were both optically and optoacoustically detected in vitro, revealing a certain variability in the photoacoustic spectra acquired by varying the excitation wavelength from 680 to 970 nm. Intramuscular engraftments of ICG‐labeled MSCs were clearly visualized by both PAI and NIRF over few days after transplantation in the hindlimb of healthy mice, suggesting that the proposed technique retains a considerable potential in the field of transplantation‐focused research and therapy. Stem cells were labeled with the Food and Drug Administration (FDA)‐approved fluorescent dye ICG, and detected by both PAI and OI, enabling to monitor the cell fate safely, in dual modality, and with good sensitivity and improved spatial resolution.      

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.     

Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Mesenchymal stem cells (MSCs) have received significant attention in recent years due to their large potential for cell therapy. Indeed, they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, several factors may restrict their use for clinical applications: the requirement of invasive procedures for their isolation, their limited numbers, and their heterogeneity according to the tissue of origin or donor. In addition, MSCs often present early signs of replicative senescence limiting their expansion in vitro, and their therapeutic capacity in vivo. Due to the clinical potential of MSCs, a considerable number of methods to differentiate induced pluripotent stem cells (iPSCs) into MSCs have emerged. iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations. Additionally, the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells. In this review, we analyze the main current protocols used to differentiate human iPSCs into MSCs, which we classify into five different categories: MSC Switch, Embryoid Body Formation, Specific Differentiation, Pathway Inhibitor, and Platelet Lysate. We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization. Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added. The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.     

Hepatogenic differentiation of mesenchymal stem cells using microfluidic chips

Directional induction and differentiation of mesenchymal stem cells (MSCs) is very important to clinical therapy, but the mechanisms that govern differentiation are not well understood. However, traditional plate culture cannot precisely control cellular behavior because cells take up substances while secreting cytokines and wastes. Here, we used a microfluidic device to culture MSCs inside a microchamber. Hepatic differentiation medium was perfused to evaluate the ability of MSCs to differentiate toward hepatic cells on the chip. Parallel differentiation on 96-well plates was used to provide a detailed comparison of the differences between the two culturing methods. After treatment for 4 weeks, differentiated cells from both groups could express hepatocyte-specific markers, including alpha-fetoprotein, tyrosine aminotransferase, and albumin. The bioactivity assays revealed that these hepatocyte-like cells could uptake lipoprotein, but cells that differentiated on the chip showed more positive signals than the cells cultured on plates. Our results indicated that a microfluidic platform might be a potential tool for cost-effective and automated cell culture, and have potential applications in reliable cell-based screens and assays.     

Pluripotency potential of human adipose-derived stem cells marked with exogenous green fluorescent protein

Musculoskeletal tissues regeneration requires rapid expansion of seeding cells both in vitro and in vivo while maintaining their multilineage differentiation ability. Human adipose-derived stem cells (ASCs) are considered to contain multipotent mesenchymal stem cells. Monolayer cultures of human ASCs were isolated from human lipoaspirates and passaged 3 times and then infected with replication-incompetent adenoviral vectors carrying green fluorescent protein (Ad/GFP) genes. Then, Ad/GFP infected human ASCs were transferred to osteogenic, chondrogenic, adipogenic, and myogenic medium. The morphological characterization of induced cells was observed using phase-contrast microscopy and fluorescence microscopy. The expression of marker proteins or genes was measured by immunocytochemical and RT-PCR analysis. Osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, aggrecan and SOX9 were positive in chondrogenic ones, peroxisome proliferator-activated receptor (PPAR-γ2) and lipoprotein lipase (LPL) were positive in adipogenic ones, and myogenin and myod1 was positive in myogenic ones. At the same time, the results of fluorescence microscopic imaging proved that the high level of GFP expression during ASCs differentiation maintained stable nearly 2 months. So the exogenous GFP and multilineage potential of human ASCs had no severe influences on each other. Since the human ASCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of GFP facilitates the research on ASCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.     

骨髓间质干细胞修复受损心肌研究进展

骨髓间充质干细胞是一种多潜能干细胞。在体外培养时,多种诱导因素可使其分化为心肌细胞等。目前进行的动物实验和临床研究表明骨髓间充质干细胞具有促进血管增生以及改善心肌梗死后心脏功能的作用,为受损心肌的治疗提供了广阔前景。但是其修复受损心肌的机制仍具有很大争议。本文就以上内容进行综述。     

Suspension-adapted Chinese hamster ovary-derived cells expressing green fluorescent protein as a screening tool for biomaterials

Synthetic biomaterials play an important role in regenerative medicine. To be effective they must support cell attachment and proliferation in addition to being non-toxic and non-immunogenic. We used a suspension-adapted Chinese hamster ovary-derived cell line expressing green fluorescent protein (GFP) to assess cell attachment and growth on synthetic biomaterials by direct measurement of GFP-specific fluorescence. To simplify operations, all cell cultivation steps were performed in orbitally-shaken, disposable containers. Comparative studies between this GFP assay and previously established cell quantification assays demonstrated that this novel approach is suitable for rapid screening of a large number of samples. Furthermore the utility of our assay system was confirmed by evaluation of cell growth on three polyvinylidene fluoride polymer scaffolds that differed in pore diameter and drawing conditions. The data presented here prove the general utility of GFP-expressing cell lines and orbital shaking technology for the screening of biomaterials for tissue engineering applications.     

Fetal vs adult mesenchymal stem cells achieve greater gene expression,but less osteoinduction

AIM: To investigate adenoviral transduction in mesenchymal stem cells(MSCs) and effects on stemness in vitro and function as a cell therapy in vivo.METHODS: Bone marrow-derived adult and fetal MSC were isolated from an equine source and expanded in monolayer tissue culture. Polyethylenimine(PEI)-mediated transfection of pc DNA3-e GFP or adenoviral transduction of green fluorescent protein(GFP) was evaluated in fetal MSCs. Adenoviral-mediated transduction was chosen for subsequent experiments. All experiments were carried out at least in triplicate unless otherwise noted. Outcome assessment was obtained by flow cytometry or immunohystochemistry and included transduction efficiency, cell viability, stemness(i.e., cell proliferation, osteogenic and chondrogenic cell differentiation), and quantification of GFP expression. Fetal and adult MSCs were then transduced with an adenoviral vector containing the gene for the bone morphogenic protein 2(BMP2). In vitro BMP2 expression was assessed by enzyme linked immunosorbent assay. In addition, MSC-mediated gene delivery of BMP2 was evaluated in vivo in an osteoinduction nude mouse quadriceps model. New bone formation was evaluated by microradiography and histology.RESULTS: PEI provided greater transfection and viability in fetal MSCs than other commercial chemical reagents. Adenoviral transduction efficiency was superior to PEI-mediated transfection of GFP in fetal MSCs(81.3% ± 1.3% vs 35.0% ± 1.6%, P < 0.05) and was similar in adult MSCs(78.1% ± 1.9%). Adenoviral transduction provided significantly greater expression of GFP in fetal than adult MSCs(7.4 ± 0.1 vs 4.4 ± 0.3 millions of mean fluorescence intensity units, P < 0.01) as well as significantly greater in vitro BMP2 expression(0.16 pg/cell-day vs 0.10 pg/cell-day, P < 0.01). Fraction of fetal MSC GFP positive cells decreased significantly faster than adult MSCs(1.15% ± 0.05% vs 11.4% ± 2.1% GFP positive at 2 wk post-transduction, P < 0.05). Cell proliferation and osteogenic differentiation in vitrowere not affected by Ad transduction in both fetal and adult MSCs, but fetal MSCs had reduced chondrogenic differentiation in vitro when compared to adult(P < 0.01). Chondrogenic differentiation was also significantly reduced in Ad-GFP transduced cells(P < 0.05). AdBMP2 transduced adult MSCs induced new bone formation in more thighs than Ad-BMP2 transduced fetal MSCs(83% vs 17% of the six treated thighs per group, P < 0.05) and resulted in increased femur midshaft diameter due to greater extent of periosteal new bone(1.57 ± 0.35 mm vs 1.27 ± 0.08 mm, P < 0.05).CONCLUSION: Fetal MSCs may be genetically manipulated ex vivo with adenoviral vectors. Nonetheless, the abbreviated expression of the exogenous gene may limit their applications in vivo.     

HO-1 modified mesenchymal stem cells modulate MMPs/TIMPs system and adverse remodeling in infarcted myocardium

The present study was to determine the effects of the heme oxygenase-1 (HO-1) modified mesenchymal stem cells (MSCs) transplantation into acute MI hearts on normalizing the ratio of MMPs/TIMPs and remodeling in infarcted myocardium. HO-1 was transfected into cultured MSCs using an adenoviral vector. 1 × 10⁶ Ad-HO-1-transfected MSCs (HO-1-MSCs) or Ad-Null-transfected MSCs (Null-MSCs) or PBS was respectively injected into rat hearts 1 h intramyocardially after myocardial infarction. The cardiac performance was significantly improved and left ventricular dilatation was significantly attenuated in HO-1-MSCs transplanted hearts. Moreover, a significant increase in microvessel density was observed in HO-1-MSCs transplanted hearts. TIMP2,3 expression in HO-1-MSCs transplanted hearts was significantly increased, and MMP2,9 expression in HO-1-MSCs transplanted hearts was significantly lower than Null-MSCs transplanted and PBS-treated hearts. TIMP1 expression did not vary significantly. Null-MSCs transplantation did not decrease the expression of MMP2,9 significantly compared with PBS-treated hearts. The ratio of TIMP2 to MMP2, and TIMP3 to MMP9 in cell-grafted hearts was increased significantly. HO-1-MSCs transplantation normalize the ratio of MMPs/TIMPs, contributing to the reversion of myocardial extracellular remodeling.     

脐带间充质干细胞——组织工程的新视角

目的 从脐带中分离培养脐带间充质干细胞(mesenchymal stem cell, MSC) 并进行鉴定,阐明其多向分化的潜在作用.方法 收集健康胎儿脐带,分离培养脐带中的间充质干细胞,以流式细胞仪对培养的间充质干细胞进行细胞表面标志检测,多种成分联合诱导其向脂肪、成骨方向分化,细胞化学染色检测诱导后的细胞变化.结果 脐带中分离培养的间充质干细胞不表达造血细胞系的标志CD34、CD45、HLA-DR,强表达CD105、CD44、CD90,在适当的诱导条件下可向脂肪及成骨方向分化.结论 脐带中存在具有多向分化潜能的间充质干细胞.     

A versatile tool for tracking the differentiation of human embryonic stem cells

The ability of human embryonic stem cells (hESCs)to undergo indefinite self-renewal in vitro and to produce lineages derived from all three embryonic germ layers both in vitro and in vivo makes such cells extremely valuable in both clinical and research settings.However,the generation of specialized cell lineages from a mixture of differentiated hESCs remains technically difficult.Tissue specific promoter-driven reporter genes are powerful tools for tracking cell types of interest in differentiated cell populations.Here,we describc the construction of modular lentivectors containing different tissue-specific promoters(Tαl of α-tubulin:αP2 of adipocyte Protein 2;and AFP of alpha fetoprotein)driving expression of humanized Renilla green fluorescent protein(hrGFP).To this end,we used MultiSite gateway technology and employed the novel vectors to successfully monitor hESC differentiation.We present a versatile method permitting target cells to bc traced.Our system will facilitate research in developmental biology,transplantation,and in vivo stem cell tracking.     

Culture and neural differentiation of rat bone marrow mesenchymal stem cells in vitro

Adult bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but can also differentiate into non-mesenchymal cells, such as neural cells, under appropriate experimental conditions. Until now, many protocols for inducing neuro-differentiation in MSCs in vitro have been reported. But due to the differences in MSCs' isolation and culture conditions, the results of previous studies lacked consistency and comparability. In this study, we induced differentiation into neural phenotype in the same MSCs population by three different treatments: beta-mercaptoethanol, serum-free medium and co-cultivation with fetal mouse brain astrocytes. In all of the three treatments, MSCs could express neural markers such as NeuN or GFAP, associating with remarkable morphological modifications. But these treatments led to neural phenotype in a non-identical manner. In serum-free medium, MSCs mainly differentiated into neuron-like cells, expressing neuronal marker NeuN, and BME can promote this process. Differently, after co-culturing with astrocytes, MSCs leaned to differentiate into GFAP(+) cells. These data confirmed that MSCs can exhibit plastic neuro-differentiational potential in vitro, depending on the protocols of inducement.     

Cell cycle dependent telomere regulation by telomerase in human bone marrow mesenchymal stem cells

Human bone marrow mesenchymal stem cells (hMSCs) are a promising source for clinical stem cell transplantation. However, telomere regulation mechanisms, as one of the possible major mechanisms by which hMSCs sustain their stem cell characteristics, remain unknown. We isolated hMSCs by plastic adhesion and characterized these cells by morphology, immune phenotype and differentiation capacity. Telomerase was found negative in hMSCs, but slightly up-regulated in hMSC-derived adipocytes by the Telomeric Repeat Amplification Protocol (TRAP) assay. Moreover, hMSCs lack the alternative lengthening of telomeres (ALT) mechanism, because the hallmarks of ALT, such as very long and heterogeneous telomeres, extra-chromosome telomere repeat DNA (ECTR), and ALT-associated promyelocytic leukemia bodies (APBs), were not evident. However, when hMSCs were arrested in S phase with a combination of serum deprivation and aphidicolin, previously undetectable telomerase activity became predominantly positive. Meanwhile, the expression level of hTERT protein and mRNA increased, paralleled with the appearance of a large cohort of synchronized hMSCs at S phase. These findings provide a profile of telomere regulation by cell cycle dependent expression of telomerase in hMSCs and may lead to a better understanding of the stem cell nature of these cells.     

Autophagy in fate determination of mesenchymal stem cells and bone remodeling

Mesenchymal stem cells (MSCs) have been widely exploited as promising candidates in clinical settings for bone repair and regeneration in view of their self-renewal capacity and multipotentiality. However, little is known about the mechanisms underlying their fate determination, which would illustrate their effectiveness in regenerative medicine. Recent evidence has shed light on a fundamental biological role of autophagy in the maintenance of the regenerative capability of MSCs and bone homeostasis. Autophagy has been implicated in provoking an immediately available cytoprotective mechanism in MSCs against stress, while dysfunction of autophagy impairs the function of MSCs, leading to imbalances of bone remodeling and a wide range of aging and degenerative bone diseases. This review aims to summarize the up-to-date knowledge about the effects of autophagy on MSC fate determination and its role as a stress adaptation response. Meanwhile, we highlight autophagy as a dynamic process and a double-edged sword to account for some discrepancies in the current research. We also discuss the contribution of autophagy to the regulation of bone cells and bone remodeling and emphasize its potential involvement in bone disease.     

Immunomodulation by mesenchymal stem cells:Interplay between mesenchymal stem cells and regulatory lymphocytes

Mesenchymal stem cells(MSCs) possess immunomodulatory properties, which confer enormous potential for clinical application. Considerable evidence revealed their efficacy on various animal models of autoimmune diseases, such as multiple sclerosis, systemic lupus erythematosus and uveitis. MSCs elicit their immunomodulatory effects by inhibiting lymphocyte activation and proliferation, forbidding the secretion of proinflammatory cytokines, limiting the function of antigen presenting cells, and inducing regulatory T(Treg) and B(Breg) cells. The induction of Treg and Breg cells is of particular interest since Treg and Breg cells have significant roles in maintaining immune tolerance. Several mechanisms have been proposed regarding to the MSCs-mediated induction of Treg and Breg cells. Accordingly, MSCs induce regulatory lymphocytes through secretion of multiple pleiotropic cytokines, cell-to-cell contact with target cells and modulation of antigen-presenting cells. Here, we summarized how MSCs induce Treg and Breg cells to provoke immunosuppression.