Optimization of use of UEA-1 magnetic beads for endothelial cell isolation

Most endothelial cells (EC) in the body belong to the microvasculature. Isolation and subsequent culture of these microvessel EC contributes greatly to our understanding of the heterogeneity and vascular specificity that exist between one organ site and another. However, a major obstacle is the overgrowth of contaminating cells (fibroblasts, pericytes, smooth-muscle cells) in cultures. Since 1990 the use of magnetic beads in combination with either a lectin, Ulex europaeus agglutinin-1 (UEA-1), or a monoclonal antibody has represented a powerful tool for the isolation/purification of microvessel EC. In the former case, operative conditions remain to be optimized to obtain pure cultures of EC.We have performed studies to optimize conditions of use for magnetic beads coated with UEA-1. Incubating beads with cells, the influences are studied of time, temperature, cell concentration, and number of beads per target cell for two cell types, human umbilical vein EC (HUVEC) and skin fibroblasts (HSF), either isolated or mixed. The effect of the last parameter was also checked on the behavior of cells undergoing proliferation after isolation. Results, expressed as isolation efficiency (from 40% to 90%) allowed us to select a 15-min incubation time at 4°C with rotary agitation, an optimal concentration of 4 x 10⁵ cells/ml, and an optimal cell:bead ration of 1:3. From a mixed cell population and in these conditions, even very low HUVEC:HSF proportions of 2.5:97.5 allowed us to obtain a pure HUVEC population in subsequent culture.Abbreviations UEA-1 Ulex europaeus agglutinin-1 - EC endothelial cells - HUVEC human umbilical vein endothelial cells - HSF human skin fibroblasts - MPC magnetic particle concentrator - IE isolation efficiency     

Isolation and characterisation of human pulmonary microvascular endothelial cells from patients with severe emphysema

Background

Loss of the pulmonary microvasculature in the pathogenesis of emphysema has been put forward as a credible alternative to the classical inflammatory cell driven proteolysis hypothesis. Mechanistic studies in this area have to date employed animal models, immortalised cell lines, primary endothelial cells isolated from large pulmonary arteries and non-pulmonary tissues and normal human pulmonary microvascular endothelial cells. Although these studies have increased our understanding of endothelial cell function, their relevance to mechanisms in emphysema is questionable. Here we report a successful technique to isolate and characterise primary cultures of pulmonary microvascular endothelial cells from individuals with severe emphysema.

Methods

A lobe of emphysematous lung tissue removed at the time of lung transplantation surgery was obtained from 14 patients with severe end-stage disease. The pleura, large airways and large blood vessels were excised and contaminating macrophages and neutrophils flushed from the peripheral lung tissue before digestion with collagenase. Endothelial cells were purified from the cell mixture via selection with CD31 and UEA-1 magnetic beads and characterised by confocal microscopy and flow cytometry.

Results

Successful isolation was achieved from 10 (71%) of 14 emphysematous lungs. Endothelial cells exhibited a classical cobblestone morphology with high expression of endothelial cell markers (CD31) and low expression of mesenchymal markers (CD90, αSMA and fibronectin). E-selectin (CD62E) was inducible in a proportion of the endothelial cells following stimulation with TNFα, confirming that these cells were of microvascular origin.

Conclusions

Emphysematous lungs removed at the time of transplantation can yield large numbers of pulmonary microvasculature endothelial cells of high purity. These cells provide a valuable research tool to investigate cellular mechanisms in the pulmonary microvasculature relevant to the pathogenesis of emphysema.     

Cell lines with developmental potential restricted to mesodermal lineages isolated from differentiating cultures of pluripotential P19 embryonal carcinoma cells

Embryonal carcinoma (EC) cells are developmentally pluripotential cells which can be induced to differentiate in cell culture to form a wide variety of cell types. To investigate the lineage relationships between cells of different types, we set out to isolate cell lines with multiple but restricted developmental potentials from differentiating cultures of P19 cells, a line of EC. By selecting for differentiated cells capable of anchorage-independent growth, we isolated cell lines which differentiated in high density cultures to form at least two cell types; myocytes that resembled fetal skeletal muscle cells and loose connective tissue cells that secreted large amounts of type I collagen. These results suggest that skeletal myocytes and connective tissue share a common precursor and that stem cells with limited but multiple developmental potentials can be isolated from differentiating cultures of P19 cells.     

Validity of a patient-derived system of tissue-specific human endothelial cells: interleukin-6 as a surrogate marker in the coronary system

The aim of our study was to evaluate the relevance of tissue- and species-specific endothelial cells (EC) to study EC-dependent mechanisms in inflammatory-mediated tissue injury. We established an isolation protocol for highly purified EC (pEC) preparations of different origin and compared EC-specific inflammatory responses. Fluorescence-activated cell separation was used to obtain pEC cultures from different human arterial (coronary artery, internal thoracic artery) and venous (umbilical vein, saphenous vein) vessels. All pEC were analyzed for growth kinetics, morphology, release of cytokines/chemokines, and expression of E-selectin. For all different EC cultures, purities of >or=99% were reproducibly achieved. The EC isolation did not affect EC growth, morphology, and function. However, characterization of pEC from different vessel materials revealed an intrinsic, tissue-specific functional heterogeneity of EC cultures. Despite an arterial and venous difference in the secretion of IL-8 and monocyte chemoattractant protein-1, especially EC from coronary arteries produced significantly more IL-6 compared with other EC types, independent of age, gender, and disease of the cell donors. In contrast, the expression of E-selectin was not affected. We conclude that the proposed isolation protocol allows the generation of a pEC bank, enabling us to study tissue-specific aspects at the level of the endothelium.     

Heterogeneity amongst splenic stromal cell lines which support dendritic cell hematopoiesis

Summary Long-term cultures (LTC) producing dendritic cells (DC) have been previously established from spleen. LTC support the development of nonadherent cells comprising small DC progenitors and immature DC. Similarly, the splenic stroma STX3, derived from a LTC which ceased DC production, can support DC development from precursors in overlaid bone marrow. The STX3 stroma is an immortalised mixed population of endothelial cells and elongated spindle-shaped cells, thought to be fibroblasts. The stromal cell components of STX3 have been studied here. A panel of 102 cell lines was established by single-cell sorting. A range of clone morphology, including cobblestone cells and elongated spindle-shaped cells, was reflective of heterogeneity in STX3. However, similar expression levels for the endothelial genes ACVRL1/ALK1, COL18A1, and MCAM in 13 splenic stromal cell lines suggested that both cell types had endothelial origin. The hematopoietic support function of stromal clones was tested in coculture assays with a bone marrow cell overlay. Splenic stromal cell lines with different morphology were both supporters and nonsupporters of hematopoiesis, in terms of foci formation or release of suspension cells. Cloning of STX3 led to the isolation of a panel of splenic endothelial cell lines heterogeneous in terms of morphology and hematopoietic support function.     

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. ¹). Human tissue samples are available only in a restricted number of laboratories or companies ^2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND ⁴ (from cerebral cortex) and cerebEND ⁵ (from cerebellar cortex), were isolated according to this procedure in the Förster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- ^4,6-9 and estrogen-treatment ¹⁰ as well as to pro-infammatory mediators, such as TNFalpha ^5,8. Moreover, we have studied the pathology of multiple sclerosis ¹¹ and hypoxia ^12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.     

Growth of cancer cell lines under stem cell-like conditions has the potential to unveil therapeutic targets

Malignant tumors comprise a small proportion of cancer-initiating cells (CIC), capable of sustaining tumor formation and growth. CIC are the main potential target for anticancer therapy. However, the identification of molecular therapeutic targets in CIC isolated from primary tumors is an extremely difficult task. Here, we show that after years of passaging under differentiating conditions, glioblastoma, mammary carcinoma, and melanoma cell lines contained a fraction of cells capable of forming spheroids upon in vitro growth under stem cell-like conditions. We found an increased expression of surface markers associated with the stem cell phenotype and of oncogenes in cell lines and clones cultured as spheroids vs. adherent cultures. Also, spheroid-forming cells displayed increased tumorigenicity and an altered pattern of chemosensitivity. Interestingly, also from single retrovirally marked clones, it was possible to isolate cells able to grow as spheroids and associated with increased tumorigenicity. Our findings indicate that short-term selection and propagation of CIC as spheroid cultures from established cancer cell lines, coupled with gene expression profiling, represents a suitable tool to study and therapeutically target CIC: the notion of which genes have been down-regulated during growth under differentiating conditions will help find CIC-associated therapeutic targets.     

Differential ability of human immunodeficiency virus isolates to productively infect human cells

Isolates of HIV showed distinct differences in the ability to replicate in continuous human hematopoietic cell lines. Moreover, although all PMC cultures obtained from healthy individuals could be infected with HIV, considerable variation in the amount of virus released from different PMC cultures was observed. These biological properties of HIV could not be correlated with clinical state, binding properties of the virus isolates to target cells, or differences in target cell CD4 antigen expression. Some isolates of HIV that could not directly infect the HUT-78 cell line showed productive infection when PMC infected with these viruses were added to this human T cell line. These observations emphasize the importance of cell to cell contact in the spread of virus. The results demonstrate for the first time the differences in the host range specificity of HIV isolates in several individual PMC cultures, and indicate that the optimal isolation of HIV is achieved with normal human PMC rather than established human cell lines.     

Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines

Three different commercially available media, known to support human and porcine-specific fibroblast cultures, were tested for their growth potential on goat skin explants. Although outgrowth of fibroblasts was observed in all media tested, irrespective of breed, porcine-specific media exhibited higher rate of growth. Using this media, three fibroblast cell lines (GSF289, GSF737, and GSF2010) from ear skin explants of normal healthy dairy goats of Kiko and Saanen breed were successfully established in culture. Liquid nitrogen stocks of these frozen cells had a viability rate of 96.2% in in vitro cultures. These cells were morphologically indistinguishable from the cell stocks prior to freezing. Analysis of the growth of a fifth passage culture revealed an ‘S’ shaped growth curve with a population doubling time of 25 h. The cell lines were found negative for microbial, fungal, and mycoplasma contaminations. These goat skin fibroblast lines and the simple method of their isolation and freezing with high rate of viability will provide additional tools to study molecular mechanisms that regulate fibroblast function and for genetic manipulation of small ruminants.     

Isolation, culture, and some morphohistochemical features of endothelial cells of the pulmonary microvasculature

Taking into account the quantitative and qualitative importance of endothelial cells of the pulmonary microvasculature (40 per cent of all lung mesenchymal cells, 26 per cent of all lung cells) the authors attempted their isolation, culture in vitro and specification of some morphohistochemical characters, using the method of Habliston et al. (1979). There were used guinea pigs and rabbits and a 0.5 and 1 per cent trypsin solution in a retrograde perfusion for the detachment of endothelial cells from their rbasement membranes. The obtained cells were maintained in culture for5 7--9 days and morphologically characterized; their caveolar enzymes were evidenced (Mg-dependent ATP-ase, acid and alkaline phosphatases, a.o.).     

SCL/tal-1-dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation

Hematopoiesis in most vertebrate species occurs in two distinct phases, primitive and definitive, which diverge from FLK1(+)VE-cadherin(-) mesoderm and FLK1(+)VE-cadherin(+) endothelial cells (EC), respectively. This study aimed at determining the stage at which hematopoietic lineage fate is determined by manipulating the SCL/tal-1 expression that is known to be essential for the early development of the primitive and definitive hematopoietic systems. We established SCL-null ES cell lines in which SCL expression is rescued by tamoxifen-inducible Cre recombinase-loxP site-mediated recombination. While no hematopoietic cells (HPC) were detected in SCL-null ES cell differentiation cultures, SCL gene reactivation from day 2 to day 4 after initiation of differentiation could rescue both primitive and definitive hematopoiesis. SCL reactivation at later phases was ineffective. Moreover, generation of VE-cadherin(+) EC that can give rise to definitive HPC required SCL reactivation prior to VE-cadherin expression. These results indicated that the competence to become HPC is acquired at the mesodermal stage by a SCL-dependent process that takes place independently of determination of endothelial fate.     

Comparison of accessory cell functions of endothelial cells and monocytes: IL-2 production by T cells and PFC generation

It has been previously shown that endothelial cells (EC) can modulate T-cell responsiveness by mimicking monocyte (AC) function in several different in vitro systems. We now report that EC and AC differ quantitatively in their ability to provide help for IL-2 generation and T-cell induced B-cell differentiation into immunoglobulin secreting cells (ISC). Equal numbers of EC were deficient when compared to AC for promoting ISC generation, but exceeded AC for IL-2 production. Adding optimal numbers of EC drive non-adherent cell cultures to produce more than twice as much IL-2 as adding any number of AC. Furthermore, small numbers of EC were capable of modulating ongoing immune responses when added to cultures containing AC. IL-2 production by PBM was doubled by the addition of enough EC to comprise only 3% of the total culture. EC do not just mimic monocytes in immune responses, but modulate these responses in unique ways.     

Establishment and biochemical characterization of tamarillo (<Emphasis Type="Italic">Solanum betaceum</Emphasis> Cav.) embryogenic cell suspension cultures

Somatic embryogenesis (SE) is an important biotechnological tool with great potential for large-scale cloning. In Solanum betaceum Cav. (tamarillo), embryogenic (EC) and non-embryogenic (non-EC) cells can be obtained from the same explant on auxin-containing medium, making this system ideal for the evaluation of biochemical changes occurring during embryogenic induction. Liquid cultures offer additional possibilities for the analysis of factors controlling SE induction, and the main objectives here were the establishment of cell suspensions and the characterization of the extracellular protein profiles in EC and non-EC cultures. Growth kinetics of liquid cultures, starting with different amounts of EC or non-EC callus or with different weight per volume ratios, were analyzed. Embryogenic suspension cultures were efficiently established starting with 40 mg of cells in 20 mL of liquid medium. Mass spectrometry and fluorometric techniques were employed to identify extracellular proteins, their hydrolytic activity, and the main classes of proteases secreted into the media of EC or non-EC cultures. Extracellular protein profiles revealed quantitative and qualitative differences between EC and non-EC suspension cultures, mainly for several hydrolytic enzymes, such as glucanases and xylanases. Proteolytic activity analysis found serine proteases, aspartic proteases, and metalloproteases in EC cultures, whereas serine proteases were dominant in non-EC lines. For the first time, a protocol for the growth of tamarillo EC and non-EC suspensions was achieved. Moreover, the comparison of protein profiles between EC and non-EC lines showed pronounced differences in the proteolytic and glycolytic enzymes secreted.     

Different characteristics of endothelial cells from central and peripheral human cornea in primary culture and after subculture

Summary Several methods for isolation and cultivation of human corneal endothelial cells have been described during the last few decades. In contrast to the situation in vivo, the cultured cells show mitogenic activity but often lose their typical morphological appearance. In this paper, we describe a technique to isolate and cultivate morphologically unchanged endothelium from the human cornea. This method revealed different characteristics of endothelial cells according to their position within the human cornea. Endothelial cells isolated from the central part have a morphology similar to that of cells in vivo (i.e., they are densely packed and show no mitogenic activity). In contrast, endothelial cells derived from the peripheral part of the cornea are characterized by mitogenic activity but their cell-to-cell attachment seems to be less tight than in vivo. The significance of these two different endothelial cell types for wound healing in the human cornea is discussed.     

A Novel Technique for Culture of Human Dermal Microvascular Endothelial Cells under either Serum-Free or Serum-Supplemented Conditions: Isolation by Panning and Stimulation with Vascular Endothelial Growth Factor

Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microvasculature require homogenous primary cultures of microvascular endothelial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contamination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonatal human foreskin by panning the cells using EN4, an anti-endothelial cell monoclonal antibody. Panned cells uniformly expressed von Willebrand factor and CD36, confirming their microvascular endothelial characteristics, whereas cells cultured without panning showed a significant degree of contamination with fibroblasts. In the presence of vascular endothelial growth factor (VEGF), HDMEC could be cultured under serum-free conditions. VEGF stimulated the growth of HDMEC in a dose-dependent manner in serum-free medium or in media supplemented with either human serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we compared the response to VEGF stimulation of HDMEC with human umbilical vein endothelial cells (HUVEC). The dose response of the two cell types to VEGF was different. This effect of VEGF on endothelial cells may be mediated by the VEGF receptorkdr,since mRNA forkdrwas detected using RT–PCR in both HDMEC and HUVEC. The procedure described in this study will make possible the culture of highly enriched HDMEC without contamination with fibroblasts and facilitate studies with these cells under defined assay conditions in a serum-free environment.     

Differential adhesive properties of sequestered asexual and sexual stages of Plasmodium falciparum on human endothelial cells are tissue independent

The protozoan parasite Plasmodium falciparum, responsible for the most severe form of malaria, is able to sequester from peripheral circulation during infection. The asexual stage parasites sequester by binding to endothelial cell receptors in the microvasculature of various organs. P. falciparum gametocytes, the developmental stages responsible for parasite transmission from humans to Anopheles mosquitoes, also spend the almost ten days necessary for their maturation sequestered away from the peripheral circulation before they are released in blood mainstream. In contrast to those of asexual parasites, the mechanisms and cellular interactions responsible for immature gametocyte sequestration are largely unexplored, and controversial evidence has been produced so far on this matter. Here we present a systematic comparison of cell binding properties of asexual stages and immature and mature gametocytes from the reference P. falciparum clone 3D7 and from a patient parasite isolate on a panel of human endothelial cells from different tissues. This analysis includes assays on human bone marrow derived endothelial cell lines (HBMEC), as this tissue has been proposed as a major site of gametocyte maturation. Our results clearly demonstrate that cell adhesion of asexual stage parasites is consistently more efficient than that, virtually undetectable of immature gametocytes, irrespectively of the endothelial cell lines used and of parasite genotypes. Importantly, immature gametocytes of both lines tested here do not show a higher binding efficiency compared to asexual stages on bone marrow derived endothelial cells, unlike previously reported in the only study on this issue. This indicates that gametocyte-host interactions in this tissue are unlikely to be mediated by the same adhesion processes to specific endothelial receptors as seen with asexual forms.     

Human capillary endothelial cells from abdominal wall adipose tissue: Isolation using an anti-pecam antibody

Summary We have developed a novel isolation technique for harvesting human capillary endothelial cells. We compared the use of eitherUlex Europaeus Agglutinin (UEA) lectin or anti-platelet endothelial cell adhesion molecule (PECAM) antibody conjugated to magnetic beads for the ability to isolate and maintain pure cultures of human capillary endothelial cells. Cells isolated using either method actively scavenged DiI-acetylated-low density lipoprotein and expressed von Willebrand factor (vWf) up to four passages as assessed by immunofluorescent labeling. Endothelial cells isolated using the anti-PECAM antibody method maintained these endothelial-specific properties for up to 12 passages while the percentage of UEA selected cells expressing these properties decreased during increasing passage number. Furthermore, while both techniques yielded cells that bind UEA at Passage six, only the antibody selected cells expressed the normal pattern of endothelial-specific cellular adhesion molecules as assessed by flow cytometry. Both cell isolates were cultured within a three-dimensional matrix of type I collagen, the antibody selected cells formed tubelike structures within 2 days, while the lectin selected cells did not. The antibody selected capillary endothelial cells were transduced with a retroviral vector containing the human growth hormone cDNA and were found to secrete growth hormone from both two- and three-dimensional cultures. We propose that anti-PECAM antibodies linked to a solid support provide a highly selective step in the isolation and maintenance of pure populations of human capillary endothelial cells from abdominal wall liposuction remnants.     

Karyotypic and phenotypic changes during in vitro aging of human endothelial cells.

Karyotypic and phenotypic changes were found in human adult endothelial cells (EC) during aging in vitro. A trisomy of chromosome 11 was found in 11 out of 12 EC cultures examined, derived from 9 cell lines from 8 donors. The incidence of this trisomy in some cell lines increased over time from 0% to as much as 100% near the end of their in vitro life span. A number of oncogenes and other important genes are on chromosome 11. These genes might play a role in the changes observed. An increase in the percentage of polyploid cells was also found near the end of the in vitro life span in 6 lines. The cellular levels of two gene products characteristic of the EC, von Willebrand factor (vWF) or Factor VIII, and angiotensin converting enzyme (ACE) were also monitored. vWf was studied in 2 lines and was decreased in both with serial passage. ACE decreased in three out of the four lines examined. These chromosomal and phenotypic changes which occur with increasing age in vitro make the endothelial cell a suitable model to study in vitro culture-related changes, senescence, cardiovascular disease, and tumorigenesis.     

Primary cultures of human umbilical chord vein endothelial cells: a biological model for studying enterococcal infection mechanisms

Although enterococcus bacteria are normal human intestinal flora, they rank as the third most common pathogen involved in hospital acquired infections. Generally, these bacteria are considered extracellular pathogens; however, an increasing number of reports indicate invasiveness to epithelial cell lines and macrophages. Despite their importance as nosocomial infection agents in patients suffering bacteremias and endocarditis, their interaction with endothelial cells has not been fully described. Herein, the nosocomial Enterococcus faecalis isolate Ef2890 from a hospitalized patient was exposed to cultured human venous endothelial cells from the umbilical chord. When the primary cell cultures were inoculated with Ef2890 and treated with bactericidal antibiotics to kill extracellular and adhered bacteria, intracellular bacteria were recovered and plated 4 h post-infection. These observations indicate that cell cultures provide a valuable biological model to study interactions between endothelium and enterococci.