Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton’s jelly

Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.     

Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue

Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary.     

Isolation and characterization of embryonic stem cell-like cells from in vitro produced goat (Capra hircus) embryos

The aim of the present study was to isolate and characterize goat embryonic stem cell-like cells from in vitro produced goat embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 150 blastocysts and 35 hatched blastocysts whereas 100 morulae were used for blastomeres isolation mechanically. The ICM derived cells or blastomeres were cultured on a feeder layer. The primary colony formation was significantly higher (P?相似文献   

Comparison between total endothelial progenitor cell isolation versus enriched Cd133+ culture

Endothelial progenitor cells (EPCs) play a role in endogenous neovascularization of ischaemic tissues. Isolation and characterization of EPCs from circulating mononuclear cells are important for developing targeted cellular therapies and reproducibility of data are the major scientific goals. Here we compared two currently employed isolation methods, i.e. from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133(+) cells, by defining the cell morphology and functional activities. We show that EPCs from cultured PBMCs resulted in an adherent population of 23% +/- 4% merged cells positive for Dil-Ac-LDL and lectin, whereas the percentage of double positive cells in cultured CD133(+) enriched cells was 50% +/- 7% (P < 0.01). These data were obtained through a novel and a more complete method of analysis of cell calculations (specifically by dividing each microscope field into 120 subfields). When stimulated with tumour necrosis factor alpha (TNF)-alpha and glucose, cell number was reduced in EPCs from total PBMCs and, more consistently, in CD133(+) enriched cells. However, both cultured total PBMCs and CD133(+) enriched cells respond similarly to TNF-alpha or glucose-induced p38-phosphorylation. EPCs from both procedures show similar results in terms of phenotype and response to modulators of their functional activities. However, when the cell phenotype of CD133(+) enrichment-derived cells was compared with that of cells from the total PBMC, a significant increase in CD133(+) expression was observed (P < 0.01) This may have relevance during intervention studies using cultured EPCs.     

Human microvessel endothelial cells: Isolation,culture and characterization

Summary Over recent years, interest in endothelial cell biology has increased dramatically with our ability to grow and study endothelial cellsin vitro. While large veins and arteries remain a quick and convenient source of endothelial cells, the great morphological, biochemical and functional heterogeneity that endothelial cells express has necessitated the development of techniques to isolate microvessel endothelial cells from different tissues to create more realisticin vitro models. The majority of isolation procedures employ selective methods to enrich microvessel endothelial cells from tissue homogenates directly, or after a period in culture. These include sieving/filtration, manual weeding, isopycnic centrifugation, selective growth media, and the use of flow cytometry or magnetic beads coupled with specific endothelial cell markers. The establishment of pure endothelial cell populations is important for studying their biochemistry and physiology and there are many morphological, immunological and biochemical criteria which can be used to characterize human endothelial cells. These range from classical markers such as von Willebrand Factor and angiotensin-converting enzyme to novel markers like platelet endothelial cell adhesion molecule-1 (CD31) and the expression of E-selectin on cytokine-activated endothelial cells.     

Spontaneous immortalization of human dermal microvascular endothelial cells

AIM: To establish and characterize a spontaneously immortalized human dermal microvascular endothelial cell line, iHDME1.METHODS: We developed a spontaneous immortalization method. This approach is based on the application of optimized culture media and culture conditions without addition of any exogenous oncogenes or carcinogens. Using this approach, we have successfully established a microvascular endothelial cell line, iHDME1, from primary human dermal microvascular endothelial cells. iHDME1 cells have been maintained in culture dishes for more than 50 passages over a period of 6 mo. Using a GFP expressing retrovirus, we generated a GFP-stable cell line (iHDME1-GFP).RESULTS: iHDME1 retain endothelial morphology and uniformly express endothelial markers such as VEGF receptor 2 and VE-cadherin but not α-smooth muscle actin (α-SM-actin) and cytokeratin 18, markers for smooth muscle cells and epithelial cells respectively. These cells retain endothelial properties, migrate in response to VEGF stimulation and form 3-D vascular structures in Matrigel, similar to the parental cells. There is no significant difference in cell cycle profile between the parental cells and iHDME1 cells. Further analysis indicates enhanced stemness in iHDME1 cells compared to parental cells. iHDME1 cells display elevated expression of CD133 and hTERT.CONCLUSION: iHDME1 cells will be a valuable source for studying angiogenesis.     

Isolation and Characterization of Embryonic Stem Cell-Like Cells From in vitro Produced Goat (Capra hircus) Embryos

The aim of the present study was to isolate and characterize goat embryonic stem cell-like cells from in vitro produced goat embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 150 blastocysts and 35 hatched blastocysts whereas 100 morulae were used for blastomeres isolation mechanically. The ICM derived cells or blastomeres were cultured on a feeder layer. The primary colony formation was significantly higher (P < 0.01) for hatched blastocysts (77.14%) than early/expanded blastocysts (54%) or morula (14%). When ICMs were isolated mechanically the primary colony formation for hatched blastocysts (90%) as well as blastocysts (66%) were significantly more than when ICMs were isolated by enzymatic digestion (60% and 30%, respectively). The colonies were disaggregated either mechanically or by enzymatic digestion for further subculture. When mechanical method was followed, the colonies remained undifferentiated up to 15 passages and three ES cell-like cell lines were produced (gES-1, gES-2, and gES-3). However, enzymatic disaggregation resulted in differentiation. The undifferentiated cells showed stem cell like morphological features, normal karyotype, and expressed stem cell specific surface markers like alkaline phosphatase, TRA-1-61, TRA-1-81, and intracellular markers Oct4, Sox2, and Nanog. Following prolonged culture of the ES cell-like cells were differentiated into several types of cells including neuron like and epithelium-like cells. In conclusion, goat embryonic stem cell-like cells can be isolated from in vitro produced goat embryos and can be maintained for long periods in culture.     

A comparison of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells

Summary A method to culture rat cerebral microvascular endothelial cells (RCMECs) was developed and adapted to concurrently obtain cultures of rat aortic endothelial cells (RAECs) without subculturing, cloning, or “weeding.” The attachment and growth requirements of endothelial cell clusters from isolated brain microvessels were first evaluated. RCMECs required fetal bovine serum to attach efficiently. Attachment and growth also depended on the matrix provided (fibronectin≈laminin>gelatin>poly-d-lysine≈Matrigel>hyaluronic acid≈plastic) and the presence of endothelial cell growth supplement and heparin in the growth medium. Non-endothelial cells are removed by allowing these cells to attach to a matrix that RCMECs attach to poorly (e.g., poly-d-lysine) and then transferring isolated endothelial cell clusters to fibronectin-coated dishes. These cell cultures, labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarboxyamine perchlorate (DiI-Ac-LDL) and analyzed using flow cytometry, were 97.7±2.6% (n=6) pure. By excluding those portions designed to isolate brain microvessels, the method was adapted to obtain RAEC cultures. RAECs do not isolate as clusters and have different morphology in culture, but respond similarly to matrices and growth medium supplements. RCMECs and RAECs have Factor VIII antigen, accumulate DiI-Ac-LDL, contain Weibel-Palade bodies, and have complex junctional structures. The activities of γ-glutamyl transferase and alkaline phosphatase were measured as a function of time in culture. RCMECs had higher enzymatic activity than RAECs. In both RCMECs and RAECs enzyme activity decreased with time in culture. The function of endothelial cells is specialized depending on its location. This culture method allows comparison of two endothelial cell cultures obtained using very similar culture conditions, and describes their initial characterization. These cultures may provide a model system to study specialized endothelial cell functions and endothelial cell differentiation. This work was funded by the National Institutes of Health grant RO1-NS-21076, and AHA-GIA 881134. Support for Ellen Gordon provided by the National Institutes of Health, NSO7144 and the Seattle Affiliate of the AHA (88-WA-111, 89-WA-112).     

A modified preplate technique for efficient isolation and proliferation of mice muscle-derived stem cells

We modified an existing protocol to develop a more efficient method to acquire and culture muscle-derived stem cells (MDSCs) and compared the characteristics of cells obtained from the two methods. This method is based on currently used multistep enzymatic digestion and preplate technique. During the replating process, we replaced the traditional medium with isolation medium to promote fibroblast-like cell adherence at initial replating step, which shortened the purifying duration by up to 4 days. Moreover, we modified the culture container to provide a stable microenvironment that promotes MDSC adherence. We compared the cell morphology, growth curve and the expression of specific markers (Sca-1, CD34, PAX7 and Desmin) between the two cell groups separately obtained from the two methods. Afterwards, we compared the neural differentiation capacity of MDSCs with other muscle-derived cell lineages. The protocol developed here is a fast and effective method to harvest and purify MDSCs from mice limb skeletal muscle.     

Isolation and properties in culture of human adrenal capillary endothelial cells

The isolation of human adrenal capillary endothelial (HACE) cells without resort to fluorescence activated cell sorting is described, together with their properties in culture. HACE cells were isolated by plating collagenase digests at high dilution in the presence of endothelial cell growth supplement, followed by clonal selection of endothelial colonies. HACE cells exhibit a typical endothelial 'cobblestone' morphology at confluence and formed 'tubes' when seeded onto 'Matrigel'. They are positive for human MHC1, and the endothelial markers ENDOCAM (CD31) and weakly CD34, they also take up dil-acetyl low density lipoprotein but are negative for Factor VIII. Their growth is strongly stimulated by FGF and inhibited by TGF-beta I. Like their much studied bovine counterparts they are robust in culture, retaining the properties described up to senescence. HACE cells provide a readily available alternative to human umbilical vein endothelial cells in that they are easily isolated pure and in quantity. They should be particularly useful in studies where human capillary, as opposed to large vessel endothelium, is required.     

Key adhesion molecules are present on long podia extended by hematopoietic cells.

BACKGROUND:We recently reported that CD34(+) hematopoietic cells and the KG1a cell line extend long, thin podia. These podia can dynamically extend and retract, often adhere to the substrate, and appear to connect cells up to 300 microm apart. The surface receptors found on these podia have not been described. METHODS:By using time-lapse fluorescent microscoscopy and immunostaining techniques, we describe a method for detecting surface receptors on these podia. This includes an in situ antibody staining procedure without fixing cells. RESULTS:We demonstrate, using CD34 selected mobilized peripheral blood cells and KG1a cells, that adhesion molecules known to play important roles in blood-cell migration and adhesion are present on these podia. These include: CD11a, CD18, CD29, CD34, CD45, CD49d, CD49e, and CD62L. Additionally, CD54 and CD44 were present on the podia extended by KG1a cells, but were not detectable on the primary CD34(+) cells. The integrin CD49d localized at the base of these podia in a time-dependent manner in KG1a cells. The frequency and morphology of these long podia on three myeloid leukemia-cell lines (KG1a, MV4-11, and AML-193) and a CD34-negative T-cell line (CEM) are also compared. KG1a and CEM cell lines extend long, dynamic podia that are similar to the podia on primary CD34(+) cells in morphology and adhesion molecule expression. The AML-193 and MV4-11 cell lines, however, did not extend these long podia. CONCLUSIONS:We describe a technique that provides a method of detecting surface receptors on thin cell membrane projections. These results support the likely role of these podia in cell migration and cell-cell communication.     

Marker profile for the evaluation of human umbilical artery smooth muscle cell quality obtained by different isolation and culture methods

Even though umbilical cord arteries are a common source of vascular smooth muscle cells, the lack of reliable marker profiles have not facilitated the isolation of human umbilical artery smooth muscle cells (HUASMC). For accurate characterization of HUASMC and cells in their environment, the expression of smooth muscle and mesenchymal markers was analyzed in umbilical cord tissue sections. The resulting marker profile was then used to evaluate the quality of HUASMC isolation and culture methods. HUASMC and perivascular-Wharton’s jelly stromal cells (pv-WJSC) showed positive staining for α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), desmin, vimentin and CD90. Anti-CD10 stained only pv-WJSC. Consequently, HUASMC could be characterized as α-SMA+ , SM-MHC+ , CD10? cells, which are additionally negative for endothelial markers (CD31 and CD34). Enzymatic isolation provided primary HUASMC batches with 90–99 % purity, yet, under standard culture conditions, contaminant CD10+ cells rapidly constituted more than 80 % of the total cell population. Contamination was mainly due to the poor adhesion of HUASMC to cell culture plates, regardless of the different protein coatings (fibronectin, collagen I or gelatin). HUASMC showed strong attachment and long-term viability only in 3D matrices. The explant isolation method achieved cultures with only 13–40 % purity with considerable contamination by CD10+ cells. CD10+ cells showed spindle-like morphology and up-regulated expression of α-SMA and SM-MHC upon culture in smooth muscle differentiation medium. Considering the high contamination risk of HUASMC cultures by CD10+ neighboring cells and their phenotypic similarities, precise characterization is mandatory to avoid misleading results.     

New method for the isolation of endothelial cells from large vessels

Background aimsNumerous protocols for the isolation of bovine aortic endothelial cells have been described in the previous literature. However, these protocols prevent researchers from obtaining the pure population of endothelial cells. Thus, this study aimed to develop a new and economical method for the isolation of pure endothelial cells by introducing a new strategy to the enzymatic digestion method proposed by previous researchers.MethodsWith the use of this method, the lumen of a bovine aorta was filled with wash medium and the outer surface of the sample was washed with alcohol for 30 seconds. Under a laminar flow hood, the inner surface of the sample was covered with filter paper. Collagenase type II was dripped onto the filter paper as a digestion enzyme. The digestion fluid was seeded in T25 flasks and fed with complete medium every 3 days.ResultsThe isolated cells were characterized by markers such as CD31, von Willebrand factor, 1,1′-dioctadecyl-1,3,3,3′,3′-tetramethylindocarbocyanine perchlorate acetylated low-density lipoprotein and angiogenesis behavior. The purity of endothelial cells was detected by flow cytometry to be of nearly 90% purity; these results were confirmed by immunostaining. Moreover, endothelial cells formed blood vessel–like tubes in a three-dimensional environment, which is specific dynamic behavior for endothelial cells.ConclusionsThe new strategy presented in the current report enables isolation of a highly pure population of endothelial cells that can survive long-term culture without inducing an overgrowth of fibroblast cells.     

山羊胚胎干细胞的分离与培养

对关中奶山羊配种后6～7天的桑椹胚和囊胚,分别采用全胚培养法、酶消化法和免疫外科法进行处理.将处理后的胚胎培养于小鼠胎儿成纤维细胞(MEF)饲养层上,分离培养山羊胚胎干细胞(Embryonic stem cell,ESC).对分离传代的山羊ESCs分别进行免疫组化染色,RT-PCR检测和体外诱导分化试验.结果表明.全胚培养法易于胚胎贴壁形成原代集落,采用全胚培养法获得的ESCs有一株目前已传至18代.山羊ESCs Nanong、Oct4、SSEA-3免疫组化染色呈阳性,SSEA-1免疫组化染色呈弱阳性,SSEA-4免疫组化染色呈阴性,RT-PCR检测显示其表达Nanog、Oct4、端粒酶、CD117.山羊ESCs经DMSO体外诱导可以向心肌细胞分化.这些试验均表明该细胞具有ESCs的生物学特性.     

CD133 immunomagnetic separation: effectiveness of the method for CD133(+) isolation from umbilical cord blood

Background aimsUmbilical cord blood (UCB) is a rich source of stem cells, the characterization and isolation of which requires specific stem cell markers and reliable and reproducible protocols.MethodsWe assessed CD133 isolation in 39 UCB samples, using a commercial immunomagnetic cell-sorting protocol, and, because of its non-reproducibility, we applied optimized protocols in an effort to improve it. These included extra-labeling of the selected CD133⁺ subpopulation and indirect labeling using anti-phycoerythrin (PE) microbeads, goat anti-mouse IgG microbeads or a combination of both. The CD34 isolation was used as a control.ResultsThe mononuclear cell fraction expressed 0.53 ± 0.06% CD133. The corresponding value for CD34 was 1.64 ± 0.15%. Following the manufacturer's instructions, the CD34 isolation resulted in a population expressing 93 ± 1.25% CD34 while, after the corresponding process, CD133⁺ expression ranged from 10% to 85% (median 60%). The optimized isolation protocols did not result in improved CD133⁺ yield. The variation in the purity of the CD133 population cannot be attributed to the different clones of CD133 used, because they do not cross-block, while other factors such as glycosylation, which could possibly interfere, do not apply in normal hematopoietic stem cells (HSC).ConclusionsCD34 isolation by the immunomagnetic method results in highly pure CD34⁺ population, while the efficient and reproducible yield of a pure CD133⁺ population is not feasible. Therefore quantification of the positive cells should follow each isolation procedure in order to confirm the number of CD133⁺ cells.     

Comparative analysis of CD1a, S-100, CD83, and CD11c human dendritic cells in normal, premalignant, and malignant tissues

A number of antibodies that recognize human dendritic cells (DC) have been identified. The main aim of this study was to compare and contrast different antigen retrieval techniques using both enzymatic and non-enzymatic treatments in order to determine the expression and distribution of several DC markers on formalin-fixed, paraffin-embedded tissues. Normal human lung, oral epithelial hyperplasia lesions, oral squamous cell carcinoma, and prostate adenocarcinoma tissues were evaluated using a panel of DC specific antibodies. The results of immunohistochemical staining for CD83, CD1a, CD11c, and S-100 DC markers were compared following the different antigen retrieval approaches. The overall best results for the analysis of tumor-associated DC were obtained with the enzymatic methods. Protease XXIV digestion was determined to be essential for detection of S-100 and CD11c positive DC, whereas trypsin and pepsin were required for the recognition of CD1a and CD83 expressing tumor-associated DC. These results could be easily adapted for routine practice and should be useful for characterization of the DC system in cancer patients for both diagnostic and prognostic purposes. In addition, standardized procedures for evaluating different subpopulations of tumor-associated DC should bring new insights in understanding of DC-tumor cell interaction.     

Up-regulation of fibrinogen-like protein 2 in porcine endothelial cells by xenogeneic CD40 signal

Acute humoral xenograft rejection (AHXR), characterized by thrombin generation and endothelial cell activation, should be overcome for the success of xenotransplantation. Fibrinogen-like protein 2 (fgl2) expressed on endothelial cells can convert prothrombin to thrombin directly, which indicates that the induced fgl2 expression in activated endothelial cells can contribute to thrombosis. In xenotransplant condition, the interaction between human CD40L and porcine endothelial CD40 can activate endothelial cells. In this study, we investigated the effect of endothelial cell activation through the interaction between human CD40L and porcine CD40 on fgl2 expression and its function as a direct prothrombinase. We found that CD40 stimulation up-regulated fgl2 expression as well as its enzymatic activity in porcine endothelial cells. Moreover, functional studies using knock-down system showed that the major factor converting human prothrombin to thrombin is fgl2 protein expressed on porcine endothelial cells. Overall, this study demonstrates that fgl2 expression can be induced by xenogeneic CD40 signal on endothelial cells and contribute to thrombin generation.     

A mechanical model for morphological response of endothelial cells under combined wall shear stress and cyclic stretch loadings

The shape and morphology of endothelial cells (ECs) lining the blood vessels are a good indicator for atheroprone and atheroprotected sites. ECs of blood vessels experience both wall shear stress (WSS) and cyclic stretch (CS). These mechanical stimuli influence the shape and morphology of ECs. A few models have been proposed for predicting the morphology of ECs under WSS or CS. In the present study, a mathematical cell population model is developed to simulate the morphology of ECs under combined WSS and CS conditions. The model considers the cytoskeletal filaments, cell–cell interactions, and cell–extracellular matrix interactions. In addition, the reorientation and polymerization of microfilaments are implemented in the model. The simulations are performed for different conditions: without mechanical stimuli, under pure WSS, under pure CS, and under combined WSS and CS. The results are represented as shape and morphology of ECs, shape index, and angle of orientation. The model is validated qualitatively and quantitatively with several experimental studies, and good agreement with experimental studies is achieved. To the best of our knowledge, it is the first model for predicting the morphology of ECs under combined WSS and CS condition. The model can be used to indicate the atheroprone regions of a patient’s artery.     

Isolation and Characterization of a Primary Proximal Tubular Epithelial Cell Model from Human Kidney by CD10/CD13 Double Labeling

Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.