Endothelial cells (ECs) are desired for their therapeutic potential in a variety of areas including gene therapy, cardiac regeneration, development of tissue-engineered vascular grafts, and prevascularized tissue transplants. Pluripotent embryonic stem cells (ESCs) can be induced to differentiate into ECs in vitro using embryoid bodies, monolayer cultures, or by genetic manipulation and immortalization. However, obtaining homogeneous cultures of proliferating ESC-derived ECs without genetic manipulation is a challenging undertaking and often requires optimization of protocols and rigorous purification techniques. Moreover, current differentiation methods that use medium containing fetal calf or bovine serum components introduce additional challenges because of our limited ability to control the differentiation signals and batch-to-batch variations of serum. We have explored the development of new medium formulations for deriving ECs from murine embryonic stem cells (mESCs) using only chemically defined reagents. We present 2 different medium formulations along with the detailed methodologies, including the optimization of extracellular matrix-derived substrates known to play a role in cell attachment and proliferation as well as cell differentiation. Characterization of the ESC-derived ECs indicate that (1) chemically defined medium formulations reproducibly generate superior ECs compared with previous serum-containing formulations, (2) fibronectin, and not collagen type-IV, is the optimal substrate for EC induction in our chemically defined medium formulations, (3) without additional activation of Notch-signaling, ESC-ECs develop predominantly into venous ECs, and (4) using these medium formulations, a second rigorous selection step is not required to generate proliferating ECs from ESCs, but it does enhance the final purity of the ECs. PMID:   21446878   PMCID:   PMC3225059   DOI:   10.1089/scd.2010.0432

Endothelial cells (EC) derived from embryonic stem cells (ESC) require additional functional characterization before they are used as a cell therapy in order to enhance their potential for engraftment and proliferation. We explore several physiologically relevant functions of ESC-derived EC (ESC-EC), such as its capacity to produce nitric oxide (NO), regulate permeability, activate and express surface molecules for the recruitment of leukocytes in response to inflammatory stimuli, migrate and grow new blood vessels, lay down extracellular matrix, and take up low-density lipoproteins. We also examined the ESC-EC ability to upregulate NO in response to shear stress and downregulate NO in response to pro-inflammatory TNF-α activation. Functional responses of ESC-EC were compared with those of cultured mouse aortic ECs. The ESC-EC exhibit most aspects of functional endothelium, but interesting differences remain. The ESC-EC produced less NO on a per cell basis, but the same amount of NO if quantified based on the area of endothelial tissue. They also exhibit increased angiogenic sprouting and are more resistant to inflammatory signals. We further characterized the subphenotype of our ESC-EC and observed both venous and arterial markers on individual cells with a larger percentage of the cells exhibiting a venous phenotype. These data support the hypothesis that the developmental default pathway is toward a venous EC, and that refinement of methods for differentiation towards arterial EC is required to maintain a homogeneous population. Copyright © 2011 S. Karger AG, Basel. PMID:   21625175   PMCID:   PMC3121553   DOI:   10.1159/000324752

Most cell culture systems grow and spread as contact-inhibited monolayers on flat culture dishes, but the embryonic stem cell (ESC) is one of the cell phenotypes that prefer to self-organize as tightly packed three-dimensional (3D) colonies. ESC also readily form 3D cell aggregates, called embryoid bodies (EB) that partially mimic the spatial and temporal processes of the developing embryo. Here, the rationale for ESC aggregatation, rather than "spreading" on gelatin-coated or mouse embryonic fibroblast (MEF)-coated dishes, is examined through the quantification of the expression levels of adhesion molecules on ESC and the calculation of the adhesive forces on ESC. Modeling each ESC as a dodecahedron, the adhesive force for each ESC-ESC binding was found to be 9.1 x 10(5) pN, whereas, the adhesive force for ESC-MEF binding was found to be an order of magnitude smaller at 7.9 x 10(4) pN. We also show that E-cadherin is the dominating molecule in the ESC-ESC adhesion and blocking E-cadherin leads to a significant reduction in colony formation. Here, we mathematically describe the preference for ESC to self-assemble into ESC-ESC aggregates and 3D colonies, rather than to bind and spread on gelatin or MEF-coated dishes, and have shown that these interactions are predominantly due to E-cadherin expression on ESC. PMID:22274712   PMCID:PMC3277780   DOI:10.4161/cam.5.6.18270

Embryoid body (EB) formation closely recapitulates early embryonic development with respect to lineage commitment. Because it is greatly affected by cell-cell and cell-substrate interactions, the ability to control the initial number of cells in the aggregates and to provide an appropriate substrate are crucial parameters for uniform EB formation. Here we report of an ultra-rapid fabrication and culture method utilizing a laser-jet printer to generate closely arrayed honeycomb microwells of tunable sizes for the induction of uniform EBs from single cell suspension. By printing various microwell patterns onto pre-stressed polystyrene sheets, and through heat induced shrinking, high aspect micromolds are generated. Notably, we achieve rounded bottom polydimethylsiloxane (PDMS) wells not easily achievable with standard microfabrication methods, but critical to achieve spherical EBs. Furthermore, by simply controlling the size of the microwells and the concentration of the cell suspension we can control the initial size of the cell aggregate, thus influencing lineage commitment. In addition, these microwells are easily adaptable and scalable to most standard well plates and easily integrated into commercial liquid handling systems to provide an inexpensive and easy high throughput compound screening platform.

Mechanical forces are important signals in the development and function of the heart and lung, growth of skin and muscle, and maintenance of cartilage and bone. The specific mechanical force "shear stress" has been implicated as playing a critical role in the physiological responses of blood vessels through endothelial cell signaling. More recently, studies have shown that shear stress can induce differentiation of stem cells toward both endothelial and bone-producing cell phenotypes. This review will highlight current data supporting the role of shear stress in stem cell fate and will propose potential mechanisms and signaling cascades for transducing shear stress into a biological signal

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious approach of pipetting an arbitrary number of cells into well plates. The interactions between the stem cells forced into close proximity of one another promotes the generation of the EBs. Because the media in each of the wells has to be manually exchanged every day, this approach is manually intensive. Moreover, because environmental parameters including cell-cell, cell-soluble factor interactions, pH, and oxygen availability can be functions of EB size, cell populations obtained from traditional hanging drops can vary dramatically even when cultured under identical conditions. Recent studies have indeed shown that the initial number of cells forming the aggregate can have significant effects on stem cell differentiation. We have developed a simple, rapid, and scalable culture method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development. Finally, these cells are easily accessible for further analysis and experimentation. This method is amenable to any lab and requires no dedicated equipment. We demonstrate this method by creating embryoid bodies using a red fluorescent mouse cell line (129S6B6-F1).

Stem cell therapies will only become clinically relevant if the stem cells differentiated in vitro function as their in vivo counterparts. Here, we employed our previously developed techniques for deriving endothelial cells (>96% purity) from mouse embryonic stem cells (ESC) and compared these with mouse aortic endothelial cells (MAEC) obtained from thoracic aortas. Immunocytochemical analysis of ESC-derived endothelial cells (EC) demonstrates that both cell types are positive for the EC markers endothelial nitric oxide synthase (eNOS), Flk-1, Flt-1, vascular endothelial cadherin (VEcad), platelet-endothelial cell adhesion molecule-1 (PECAM-1), and CD34. However, ESC-derived EC express slightly lower levels of PECAM-1 and VE-cadherin, and significantly lower levels of acetylated low-density lipoprotein (LDL) uptake and von Willebrand factor. Although ESC-derived EC do express VE-cadherin, the VE-cadherin in the ESC-derived EC did not localize as well at the cell-cell junctions as in the MAEC. Interestingly, ESC-derived EC express much greater levels of the endothelial and hematopoietic stem cell marker CD34 and vasculogenic and angiogenic sprouting than MAEC. These results indicate that ESC-derived EC share some key characteristics of ’mature’ EC, while retaining markers of alternate phenotypes including immature endothelium. Copyright (c) 2006 S. Karger AG, Basel.

Vascular progenitor cells derived from stem cells could potentially lead to a variety of clinically relevant applications including cell-based therapies and tissue engineering. Here we describe methods for isolating purified proliferating populations of vascular endothelial cells from mouse embryonic stem cells (mESC) using Flk-1 positive sorted cells, VEGF supplementation, and a rigorous manual selection technique required for endothelial cell purification and expansion. Using this in vitro derivation procedure, it is possible to obtained millions of cells at various stages of differentiation, with the potential for up to 25 population doublings.

Magnetic cell separation has become a popular technique to enrich or deplete cells of interest from a heterogeneous cell population. One important aspect of magnetic cell separation is the degree to which a cell binds paramagnetic material. It is this paramagnetic material that imparts a positive magnetophoretic mobility to the target cell, thus allowing effective cell separation. A mathematical relationship has been developed to correlate magnetic labeling to the magnetophoretic mobility of an immunomagnetically labeled cell. Four parameters have been identified that significantly affect magnetophoretic mobility of an immunomagnetically labeled cell: the antibody binding capacity (ABC) of a cell population, the secondary antibody amplification (ψ), the particle-magnetic field interaction parameter (ΔχVm), and the cell diameter (Dc). The ranges of these parameters are calculated and presented along with how the parameters affect the minimum and maximum range of magnetophoretic mobility. A detailed understanding of these parameters allows predictions of cellular magnetophoretic mobilities and provides control of cell mobility through selection of antibodies and magnetic particle conjugates.