In vitro endothelial potential of human UC blood-derived mesenchymal stem cells

BACKGROUND: Human mesenchymal stem cells (MSC) possess powerful ex vivo expansion and versatile differentiation potential, placing themselves at the forefront of the field of stem cell-based therapy and transplantation. Of high clinical relevance is the endothelial differentiation potential of MSC, which can be used to treat various forms of ischemic vascular disease. METHODS: We investigated whether human umbilical cord blood (UCB)-derived MSC are able to differentiate in vitro along an endothelial lineage, by using flow cytometry, RT-PCR and immunofluorescence analyzes, as well as an Ab array method. RESULTS: When the cells were incubated for up to 3 weeks in the presence of VEGF, EGF and hydrocortisone, they began to express a variety of endothelial lineage surface markers, such as Flk-1, Flt-1, VE-Cadherin, vWF, VCAM-1, Tie-1 and Tie-2, and to secrete a specific set of cytokines. Differentiated cells were also found to be able to uptake low-density lipoprotein and form a tubular network structure. DISCUSSION: These observations have led us to conclude that UCB-derived MSC retain endothelial potential that is suitable for basic and clinical studies aimed at the development of vasculature-directed regenerative medicine.     

Phenotypic and proliferative modulation of human mesenchymal stem cells via crosstalk with endothelial cells

The purpose of this work was to investigate if a coculture system of human mesenchymal stem cells (hMSC) with endothelial cells (human umbilical vein endothelial cells, HUVEC) could modulate the phenotype and proliferation of harvested MSCs. In addition to previous investigations on the crosstalk between these two cell types, in the present work different relative cell ratios were analyzed for long, therapeutically relevant, culture periods. Moreover, MSCs osteogenic commitment was assessed in a non-osteogenic medium and in the presence of HUVECs through magnetic cell separation, cell quantification by flow cytometry, morphology by fluorescent microscopy, metabolic activity and gene expression of osteogenic markers. Collectively, the present findings demonstrate that, by coculturing MSCs with HUVECs, there was not only the promotion of osteogenic differentiation (and its enhancement, depending on the relative cell ratios used), but also a significant increase on MSCs proliferation. This augmentation in cell proliferation occurred independently of relative cell ratios, but was favored by higher relative amounts of HUVECs. Taken together, this data suggests that HUVECs not only modulate MSC phenotype but also their proliferation rate. Therefore, a coculture system of MSCs and HUVECs can a have a broad impact on bone tissue engineering approaches.     

人骨髓间充质干细胞向血管内皮细胞诱导分化的研究

目的:分析人骨髓间充质干细胞(hMSCs)和脐静脉内皮细胞(hUVECs)的基因表达差异,探讨体外基因转染诱导内皮分化的可行性以及作为血管组织工程种子细胞来源的应用前景。方法:分别从人骨髓和脐静脉分离间充质干细胞(hMSCs)和内皮细胞(hUVECs),扩增培养后进行流式细胞仪、免疫细胞化学,免疫荧光鉴定和超微结构观察。通过BiostarH-40S表达谱芯片分析,选择两者的差异表达基因,导入hMSCs,经RT-PCR、ELISA鉴定该基因的转染和表达,并分析hMSCs的内皮分化程度。结果:hMSCs表达内皮细胞的多种特异性mRNA,经VEGFl65基因瞬时转染后RT-PCR有明显条带,ELISA定量检测VEGF165蛋白表达为(707.9±11.3)ng/L,同时CD44表达明显下调38.80%,CD31则明显上调达56.82%,FI-1,FVⅢAg和CD34的表达也有不同程度升高。结论:hMSCs具有内皮分化潜能,体外基因转染诱导hMSCs产生功能性内皮细胞和组织工程化血管具有广阔前景。     

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child’s birth; however, its importance as a “store house” of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton’s jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.     

Molecular and ultrastructural characterization of endothelial cells differentiated from human bone marrow mesenchymal stem cells

In this study characterization of endothelial cells differentiated from human bone marrow mesenchymal stem cells (hBMCs) was investigated in relation to their capillary network formation potential. Differentiation was performed in presence of vascular endothelial growth factor (VEGF) and insulin like growth factor-1 (IGF-1). A panel of cellular and molecular markers was used for characterization of the endothelial cells. The cells were strongly positive for von Willebrand factor (vWF) and vascular endothelial growth factor receptor 2 (VEGFR2) when measured at protein and mRNA levels. Development of endothelial cells was found to be associated with formation of typical organelles such as Weibel Palade (WP) bodies, Cavealae and pinocytic vesicles. Early vessel growth was also evidenced by showing specific junctions between the cells. The migratory and angiogenic properties of the cells were confirmed by showing capillary network formation in vitro. These results indicate that the capacity of endothelial cells differentiated from hBMSCs in formation of vascular system is consistent with molecular and structural development.     

Separation and characterization of human T lymphocytes with varying adhesiveness for endothelial cells

Previous studies in this laboratory have demonstrated that the adhesion of T lymphocytes to endothelial cell (EC) monolayers in vitro can be increased by preincubation of the EC with interferon-gamma, interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF), or lipopolysaccharide (LPS), or by stimulation of the T cells with phorbol esters. In this report, we have demonstrated that three subpopulations of human peripheral blood T cells can be identified on the basis of their abilities to bind to EC: (1) a strongly binding group which binds to unstimulated EC; (2) an intermediately binding subset which adheres to EC only if these cells have been stimulated with IL-1, TNF, or LPS; and (3) a weakly binding subpopulation which adheres poorly to either unstimulated or stimulated EC. The more adhesive subgroups had larger cellular volumes than the less adhesive cells, were relatively enriched in cells bearing the OKM1 surface marker, and expressed relatively greater amounts of the lymphocyte-function-associated-1 molecule. Stimulation of the EC to bind increased numbers of T cells by IL-1, TNF, and LPS appeared to be mediated by the expression of a common adhesion molecule on the EC.     

IL-4 increases human endothelial cell adhesiveness for T cells but not for neutrophils

The adhesion of leukocytes to vascular endothelium is the first step in their passage from the blood into inflammatory tissues. By modulating endothelial cell (EC) adhesiveness for leukocytes, cytokines may regulate leukocyte accumulation and hence the nature and progression of inflammatory responses. We have found that the T cell cytokine IL-4 increases the adhesion of T cells, but not neutrophils, to human umbilical vein EC monolayers. The increase in T cell adhesion induced by IL-4 was dose dependent (ED50 = 5 U/ml) and peaked around 33 U/ml. No increase in adhesion of neutrophils was observed at concentrations of IL-4 up to 1000 U/ml. The kinetic of the increase in T cell adhesion exhibited a steady rise peaking between 18 and 24 h before returning to basal levels by 72 h. The IL-4 specificity of the effect was confirmed by the ability of neutralizing anti-IL-4, but not anti-TNF, antibodies to abolish the effect. The increase in T cell-EC adhesion was due to an effect of IL-4 on EC inasmuch as preincubation of the T cells with IL-4 did not increase T cell binding. Furthermore, preincubation of A549 epithelial cell line monolayers with IL-4 caused no increase in T cell binding whereas A549 cells and EC showed a similarly enhanced adhesiveness for T cells after preincubation with IL-1, TNF, or IFN-gamma. EC treated with IL-4 retained their increased adhesiveness for T cells after light fixation, suggesting that IL-4 up-regulates binding by increasing the expression or accessibility of EC surface receptors for lymphocytes. Although antibodies to intercellular adhesion molecule-1 (CD54) and the beta-chain (CD18) of lymphocyte function-associated Ag-1 (CD11a/CD18) partially inhibited T cell adhesion to unstimulated EC, they did not affect the increase in adhesion due to IL-4 stimulation, indicating that the increased binding resulted from the generation of an alternative binding receptor(s) on the EC membrane. These findings suggest that IL-4 may play a role in the selective recruitment of T cells into sites of immune-mediated chronic inflammation.     

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.     

Thrombin-induced adherence of neutrophils to cultured endothelial monolayers: increased endothelial adhesiveness

We examined the effects of alpha-thrombin on the adherence of neutrophils to endothelial cell monolayers. Endothelial cells derived from the ovine pulmonary artery and ovine neutrophils were used. Thrombin (10(-8) M) resulted in a time-dependent increase in neutrophil adherence to the endothelium. The response was concentration-dependent with a maximal response at 10(-8) M. Thrombin did not induce neutrophil adherence either to plastic or to endothelial cell-derived matrix. The adherence response was inhibited in the presence of alpha-thrombin that had been inactivated with anti-thrombin III (1U:1U) or with hirudin (1 U/ml). However, the addition of either anti-thrombin III or hirudin simultaneously with alpha-thrombin to the cultured endothelial monolayers did not prevent neutrophil adherence. The monoclonal antibody MoAb 60.3, which precipitates a complex of four neutrophil surface glycoproteins (CDw18) was used to further characterize the reaction. MoAb 60.3 decreased the thrombin-induced adherence of neutrophils to the endothelial monolayer. Addition of 10(-8) M thrombin to the endothelial monolayer for 60 min, followed by washing the endothelium with fresh medium, caused resting neutrophils to adhere to the endothelial monolayers. MoAb 60.3 decreased neutrophil adherence to the washed endothelium. The factor(s) responsible for adherence was partially transferable. Medium obtained from incubating endothelial monolayers with thrombin (10(-8) M) for 60 min, adding hirudin to the medium to inactivate thrombin, and transferring it to untreated endothelial monolayers, elicited neutrophil adherence. The response was less than that obtained with thrombin alone (22.9 +/- 2.3% vs. 12.9 +/- 3.3%). The results indicate that the catalytic site of the thrombin molecule is responsible for the adherent activity. Thrombin elicits a rapid activation of endothelial cells with a response that involves the expression of endothelial adhesion sites and sites that interact with the neutrophil CDw18 adhesive glycoprotein complex. In addition, soluble transferable factor(s) which are generated by the endothelium also contribute to thrombin-induced neutrophil adherence.     

Ammonia plasma treatment of polystyrene surfaces enhances proliferation of primary human mesenchymal stem cells and human endothelial cells

The control of surface properties is a substantial step in the development and improvement of biomaterials for clinical applications as well as for their use in tissue engineering. Interaction of the substrate surface with the biochemical or biological environment is crucial for the outcome of the applied biomaterial and therefore should meet specific requirements regarding the chemical composition, wettability, elasticity, and charge. In this study, we examined the effect of chemical groups introduced by low pressure plasma treatments of polystyrene surfaces on the cell behavior of primary human mesenchymal stem cells (hMSCs) and dermal microvascular endothelial cells (hDMECs). X-ray photoelectron spectroscopy analysis and contact angle measurements were employed to evaluate ammonia-, carbon dioxide-, and acrylic acid-plasma modifications to substrate surfaces. HMSCs and hDMECs were analyzed simultaneously to identify the most suitable surface functionalization for each cell type. Significantly higher cell proliferation was detected on ammonia plasma-treated surfaces. Cell-material interaction could be shown on all created interfaces as well as the expression of typical cell markers. Hence, the applied plasma treatment presents a suitable tool to improve culture condition on polystyrene for two important cell types (hMSCs and hDMECs) in the field of tissue engineering.     

Growth factor-defined culture medium for human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) are potential cellular sources of therapeutic stem cells as they have the ability to proliferate and differentiate into a wide array of mesenchymal cell types such as osteoblasts, chondroblasts and adipocytes. hMSCs have been used clinically to treat patients with graft vs. host disease, osteogenesis imperfect, or alveolar cleft, suggesting that transplantation of hMSCs is comparatively safe as a stem cell-based therapy. However, conventional culture medium for hMSCs contains fetal bovine serum (FBS). In the present study, we developed a growth factor-defined, serum-free medium for culturing hMSCs. Under these conditions, TGF-beta1 promoted proliferation of hMSCs. The expanded hMSC population expressed the human pluripotency markers SSEA-3, -4, NANOG, OCT3/4 and SOX2. Furthermore, double positive cells for SSEA-3 and a mesenchymal cell marker, CD105, were detected in the population. The potential to differentiate into osteoblasts and adipocytes was confirmed. This work provides a useful tool to understand the basic biological properties of hMSCs in culture.     

A simple protocol for transfecting human mesenchymal stem cells

Objectives and results

Mesenchymal stromal cells (MSCs) are 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 have significant expansion capability. These characteristics make them strong candidates for delivering genes and restoring organ systems function. However, as other primary cells, MSCs are difficult to transfect. In order to standardize a simple protocol for transfection of MSCs, we conducted a series of experiments and achieved a protocol that does not require the use of viral particles or specific expensive equipment.

Conclusion

MSCs transfection at early passages using a ratio lipid/DNA of 3.0 µL/µg with Lipofectamine 3000^® yields good transfection efficiencies for human MSCs (up to 26%) and is rapid, simple, and safe.

     

Viscoelastic behaviour of human mesenchymal stem cells

Background  

In this study, we have investigated the viscoelastic behaviour of individual human adult bone marrow-derived mesenchymal stem cells (hMSCs) and the role of F-actin filaments in maintaining these properties, using micropipette aspiration technique together with a standard linear viscoelastic solid model.     

Efficient in vitro myogenic reprogramming of human primary mesenchymal stem cells and endothelial cells by Myf5

Background information. The identification of a source of stem cells able to regenerate skeletal muscle was the goal of numerous studies with the aim to develop new therapeutic approaches for genetic muscle diseases or muscle injuries. A series of studies have demonstrated that stem cells derived from various tissues may have a role in the regeneration of damaged muscles, but this contribution is always very weak. Thus we established a project aiming to reprogramme non‐muscle cells into the skeletal striated differentiation pathway. Results. We transduced several human primary adult stem or progenitor cells using a recombinant lentivirus containing the coding sequence of the Myf5 gene considered as a master gene for the determination of skeletal striated muscle. These original results are the first demonstration of a myogenic conversion of human mesenchymal and endothelial cells by Myf5. Conclusions. The procedure described in the present paper could be used to develop new research protocols with the prospect of using these cells as therapeutic agents.     

间充质干细胞分化为内皮细胞的意义和细胞学基础

间充质干细胞(mesenchymalstemcells,MSCs)主要存在于骨髓中,是多潜能干细胞,在脐血、外周血、脂肪、皮肤等多种组织中也相继分离出MSCs。MSCs具有独特的免疫特性,在异种异体环境内长期存在,使其临床应用前景更为广泛。目前,MSCs的分离培养、诱导分化及鉴定体系已趋成熟,理论上可分化为所有中胚层来源的细胞,内皮细胞来源于中胚层,因此MSCs具有分化为内皮细胞的可能性。本文对MSCs内皮分化意义和细胞学基础及其新近的研究进展作一综述。     

Priming human adipose-derived mesenchymal stem cells for corneal surface regeneration

Limbal stem cells (LSC) maintain the transparency of the corneal epithelium. Chemical burns lead the loss of LSC inducing an up-regulation of pro-inflammatory and pro-angiogenic factors, triggering corneal neovascularization and blindness. Adipose tissue-derived mesenchymal stem cells (AT-MSC) have shown promise in animal models to treat LSC deficiency (LSCD), but there are not studies showing their efficacy when primed with different media before transplantation. We cultured AT-MSC with standard medium and media used to culture LSC for clinical application. We demonstrated that different media changed the AT-MSC paracrine secretion showing different paracrine effector functions in an in vivo model of chemical burn and in response to a novel in vitro model of corneal inflammation by alkali induction. Treatment of LSCD with AT-MSC changed the angiogenic and inflammatory cytokine profile of mice corneas. AT-MSC cultured with the medium that improved their cytokine secretion, enhanced the anti-angiogenic and anti-inflammatory profile of the treated corneas. Those corneas also presented better outcome in terms of corneal transparency, neovascularization and histologic reconstruction. Priming human AT-MSC with LSC specific medium can potentiate their ability to improve corneal wound healing, decrease neovascularization and inflammation modulating paracrine effector functions in an in vivo optimized rat model of LSCD.     

De-differentiation-derived mesenchymal stem cells demonstrate selective repression in H19 bioregulatory RNA gene expression

Cellular de-differentiation can induce anticancer activity that makes cells resistant to carcinogenesis, but the molecular mechanism of this phenomenon has not been defined. To determine whether stable molecular changes develop in association with the process of de-differentiation, DNA microarray analyses were performed. These analyses compared control undifferentiated cells with three carcinogenesis-resistant clones of de-differentiated cells that were derived from mature adipocytes. The results of analysis of 6,000 genes and 6,000 ESTs establish that relative to control cells, all three de-differentiation-derived cell clones demonstrate that only one gene shows a consistent difference in expression. The expression of the H19 bioregulatory RNA is repressed an average of >fourfold in all de-differentiated cell clones. Real-time PCR analyses confirm these findings. This suggests that decreased H19 expression may account, at least in part, for the anticancer activity observed in de-differentiated cell clones.