Pure Population Of Endothelial Cells Research Articles

Monolayer Generation of Vascular Endothelial Cells from Human Pluripotent Stem Cells.

The use of human pluripotent stem cells (hPSCs) for modeling human diseases and therapeutic applications requires differentiation methods that generate physiologically relevant cell types in a robust and standardized way. Herein, we describe an efficient and scalable monolayer protocol to convert pluripotent stem cells into vascular endothelial cells using defined culture conditions.The combinatorial use of small molecule compounds, growth factors as well as morphogens directs human pluripotent stem cells toward endothelial cells within 6days. The protocol has the capacity to generate endothelial cells with high efficiencies of up to 80%. An additional immunomagnetic cell purification step that is based on the surface marker VE-cadherin results in a virtually pure population of endothelial cells. In a subsequent expansion step human PSC-derived endothelial cells can be further propagated, while maintaining their endothelial identity. Thus, our differentiation protocol enables the generation of hPSC-derived endothelial cells at a scale that is relevant for drug discovery campaigns or clinical applications.

Highly Efficient Differentiation of Endothelial Cells from Pluripotent Stem Cells Requires the MAPK and the PI3K Pathways.

Pluripotent stem cells are a promising source of endothelial cells (ECs) for the treatment of vascular diseases. We have developed a robust protocol to differentiate human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into ECs with high purities (94%-97% CD31+ and 78%-83% VE-cadherin+ ) in 8 days without cell sorting. Passaging of these cells yielded a nearly pure population of ECs (99% of CD31+ and 96.8% VE-cadherin+ ). These ECs also expressed other endothelial markers vWF, Tie2, NOS3, and exhibited functions of ECs such as uptake of Dil-acetylated low-density lipoprotein and formation of tubes in vitro or vessels in vivo on matrigel. We found that FGF2, VEGF, and BMP4 synergistically induced early vascular progenitors (VPs) from hiPSC-derived mesodermal cells. The MAPK and PI3K pathways are crucial not only for the initial commitment to vascular lineages but also for the differentiation of vascular progenitors to ECs, most likely through regulation of the ETS family transcription factors, ERG and FLI1. We revealed novel roles of the p38 and JNK MAPK pathways on EC differentiation. Furthermore, inhibition of the ERK pathway markedly promoted the differentiation of smooth muscle cells. Finally, we demonstrate that pluripotent stem cell-derived ECs are capable of forming patent blood vessels that were connected to the host vasculature in the ischemic limbs of immune deficient mice. Thus, we demonstrate that ECs can be efficiently derived from hiPSCs and hESCs, and have great potential for vascular therapy as well as for mechanistic studies of EC differentiation. Stem Cells 2017;35:909-919.

Directing human embryonic stem cells towards functional endothelial cells easily and without purification.

Hemangioblasts or blood islands only arise in early development thereby the sources to obtain these bi-potential cells are limited. While previous studies have isolated both lineages in vitro through the hemangioblast, derivation efficiency was rather low due to cellular damage attributed by enzyme usage and fluorescent activated cell sorting (FACS). This study focused on avoiding the use of damaging factors in the derivation of endothelial cells (ECs). Single cell H9-human embryonic stem cells (hESCs) were obtained by using a mild dissociation protocol then human embryoid body (hEB) formation was performed under hemangioblast differentiation conditions. The hEBs were subjected to a two-stage cytokine treatment procedure. Subsequent culture of the adhesive cells in day 4 hEBs gave arise to a seemingly pure population of ECs. The hESC-derived ECs were characterized by identifying signature endothelial gene and protein markers as well as testing for in vitro functionality. Furthermore, in vivo functionality was also confirmed by transplanting the cells in hindlimb ischemic murine models. We demonstrate that the genetic change required for EC derivation precedes blast colony formation. Furthermore, cell damage was prevented by abating enzyme usage and FACS, resulting in a high yield of ECs upon adhesion. Under this method, confluent cultures of ECs were obtainable 4 days after hEB formation which is significantly faster than previous protocols.

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.

Mouse Models and Techniques for the Isolation of the Diabetic Endothelium

Understanding the molecular mechanisms underlying diabetic endothelial dysfunction is necessary in order to improve the cardiovascular health of diabetic patients. Previously, we described an in vivo, murine model of insulin resistance induced by feeding a high-fat diet (HFD) whereby the endothelium may be isolated by fluorescence-activated cell sorting (FACS) based on Tie2-GFP expression and cell-surface staining. Here, we apply this model to two new strains of mice, ScN/Tie2-GFP and ApoE(−/−)/Tie2-GFP, and describe their metabolic responses and endothelial isolation. ScN/Tie2-GFP mice, which lack a functional toll-like receptor 4 (TLR4), display lower fasting glucose and insulin levels and improved glucose tolerance compared to Tie2-GFP mice, suggesting that TLR4 deficiency decreases susceptibility to the development of insulin resistance. ApoE(−/−)/Tie2-GFP mice display elevated glucose and cholesterol levels versus Tie2-GFP mice. Endothelial isolation by FACS achieves a pure population of endothelial cells that retain GFP fluorescence and endothelial functions. Transcriptional analysis of the aortic and muscle endothelium isolated from ApoE(−/−)/Tie2-GFP mice reveals a reduced endothelial response to HFD compared to Tie2-GFP mice, perhaps resulting from preexisting endothelial dysfunction in the hypercholesterolemic state. These mouse models and endothelial isolation techniques are valuable for assessing diabetic endothelial dysfunction and vascular responses in vivo.

Characterizing Endothelial Cells Derived from the Murine Embryonic Stem Cell Line CCE

Embryonic stem cells (ESC) are defined by two main properties of self-renewal and their multipotency to differentiate into virtually all cell types of the body, including endothelial cells. ESCs have been widely regarded as an unlimited source of cells in regeneration medicine and also an ideal in vitro model to investigate complex developmental processes. Here, we report a simple and efficient in vitro model to derive a nearly pure population of endothelial cells from a murine ESC line. CCE ES cells are exposed to alpha-MEM medium containing 10% FBS for 4 days and then cultured in endothelial basal-2 medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), and 2% FBS for 42 days. The cells acquired a relatively uniform endothelial cell morphology and were able to propagate and expand in culture. When murine ES cell-derived endothelial cells (MESDECs) were cultured on Matrigel and incubated for 48 h, vessel-like tube structures consisting of CD31 (PECAM-1) or BS-1 immunoreactive cells were developed. Immunocytochemistry and RT-PCR analyses revealed that MESDECs express endothelial cell-specific marker proteins such as Flk-1, PECAM-1, Tie-1, and Tie-2, in which the expressions persist for long periods of time after differentiation. The cells were also capable of taking up acetylated low-density lipoprotein (LDL) in culture. Our data suggest that MESDECs could provide a suitable in vitro model to study molecular events involved in vascular development and open up a new therapeutic strategy in regeneration medicine of cardiovascular disorders.

Tumor-associated endothelial cells display GSTP1 and RARβ2 promoter methylation in human prostate cancer

BackgroundA functional blood supply is essential for tumor growth and proliferation. However, the mechanism of blood vessel recruitment to the tumor is still poorly understood. Ideally, a thorough molecular assessment of blood vessel cells would be critical in our comprehension of this process. Yet, to date, there is little known about the molecular makeup of the endothelial cells of tumor-associated blood vessels, due in part to the difficulty of isolating a pure population of endothelial cells from the heterogeneous tissue environment.MethodsHere we describe the use of a recently developed technique, Expression Microdissection, to isolate endothelial cells from the tumor microenvironment. The methylation status of the dissected samples was evaluated for GSTP1 and RARβ2 promoters via the QMS-PCR method.ResultsComparing GSTP1 and RARβ2 promoter methylation data, we show that 100% and 88% methylation is detected, respectively, in the tumor areas, both in epithelium and endothelium. Little to no methylation is observed in non-tumor tissue areas.ConclusionWe applied an accurate microdissection technique to isolate endothelial cells from tissues, enabling DNA analysis such as promoter methylation status. The observations suggest that epigenetic alterations may play a role in determining the phenotype of tumor-associated vasculature.

Isolation and culture of microvascular endothelial cells from the primate corpus luteum.

Endothelial cells have common as well as specialized roles in different tissues and organs; as such, the abundant endothelial cells in the corpus luteum (> 50% of total cell population) could have unique activities necessary for luteal function. Our objective was to establish a method for isolating a pure population of endothelial cells from the primate corpus luteum and to determine the basic conditions for in vitro culture. Corpora lutea collected from rhesus monkeys throughout the luteal phase of the menstrual cycle were minced and enzymatically dispersed into single cell suspensions. Endothelial cells were isolated from the remaining cells (i.e., steroidogenic, fibroblastic, etc.) utilizing magnetic beads labeled with a lectin, Ulex europaeus agglutinin-1 (UEA-1), which binds a sugar found only on primate endothelial cells. After exposure to a magnetic field, UEA-1-negative (-) cells were decanted from the pelleted UEA-1-positive (+) cells; to remove beads from the UEA-1 (+) cells, excess sugar was applied. After optimization of the bead-to-cell ratio, the UEA-1 (+) group contained a population of cells 8-12 microns in diameter (typical endothelial cell size) and < 1% steroidogenic cells. UEA-1 (-) cells were larger (15-35 microns) and stained histochemically for the steroidogenic marker, 3 beta-hydroxysteroid dehydrogenase. Immunocytochemical analysis demonstrated that > 93% of all cells in the UEA-1 (+) group stained positive for the specific endothelial cell marker, platelet/endothelial cell adhesion molecule-1. Cultured UEA-1 (+) cells produced low levels of progesterone and were unresponsive to hCG (100 ng/ml). In contrast, cultured UEA-1 (-) and mixed (unsorted) cells produced high basal levels of progesterone and exhibited a > 3-fold increase in response to hCG treatment. Preliminary experiments comparing different culture media and matrices demonstrated that 1) cell proliferation was unaffected by type of medium (i.e., Dulbecco's Modified Eagle Medium [DMEM]/ F12 or McCoy's 5A); 2) the presence of serum was essential in the absence of added growth factors, and 3) extracellular matrix had a profound effect on proliferation. UEA-1 (+) cells exhibited a dose-dependent increase in cell proliferation in response to vascular endothelial growth factor (VEGF) in the presence and absence of fetal calf serum. In the absence of serum, VEGF stimulated proliferation of UEA-1 (+) cells plated on fibronectin but not collagen I, whereas in the presence of 10% fetal calf serum, both matrices supported VEGF-induced mitogenesis. These studies provide for the first time an efficient, reliable method for isolating primate luteal endothelial cells and describe in vitro culture conditions for subsequent studies examining luteal endothelial cell function and regulation.

Isolation and characterization of an established endothelial cell line from transgenic mouse hemangiomas

A murine endothelial cell line was isolated from hemangiomas induced by expression of the polyoma early region gene in transgenic mice. After two cell sortings using acetylated low-density lipoprotein with a fluorescent label (Dil-Ac-LDL), a pure population of endothelial cells has been carried for more than 60 passages from the animal. The cells retain endothelial cell properties such as a characteristic cobblestone appearance at confluency, contact-inhibited growth, and active uptake of Ac-LDL. Expression analysis shows that the cells express both the polyoma transgene and the von Willebrand factor, an endothelial cell marker. Subcutaneous injection of the cultured endothelial cells into nontransgenic histocompatible mice or nude mice led to hemangioma formation, and endothelial cells were reisolated by cell sorting from these secondary hemangiomas. This cell line represents a renewable source of murine endothelial cells derived from transgenic mice that can be studied both in vitro and by reintroduction into a host.