Differentiation of cells within EBs is directed by morphogenic cues comprising the intercellular and surrounding extracellular microenvironment, including exogenously administered molecules and endogenous factors produced by the ESCs. Individual aspects of the microenvironment can be studied rather simply in planar culture formats, but similar to a developing embryo, the 3D organization of an EB is inherently comprised of a complex milieu of integrated signals that synergistically affect cell differentiation. Although the 3D assembly of cells to form EBs presents unique challenges for regulating the homogeneity of stem cell differentiation, attempts to control EB size, soluble factor delivery, extracellular matrix (ECM) interactions and cell-cell adhesions within EBs may influence differentiated cell phenotypes ().
2.1. Size Control
The size of EBs, typically in the range of 100–400 µm, is thought to be a simple, yet important physical parameter capable of influencing the proportion of cells differentiating toward different lineages. EB size, which is primarily a function of the number of ESCs constituting each cell aggregate, impacts other environmental parameters affecting differentiation, such as the diffusion of soluble molecules and the extent of ECM-cell and cell-cell adhesive interactions. Recent developments in EB formation techniques have enabled more controlled systems capable of modulating EB size in order to begin to determine the effects on subsequent differentiation of the cells.
As described above, forced aggregation of ESCs using multi-well round-bottomed plates or microtechnologies provides a very direct manner to precisely control the number of cells in individual cell aggregates. For example, the number of cells used to form hanging drops can influence the chondrogenic differentiation potential of EBs (59
). Likewise, forced centrifugation studies examining hematopoietic differentiation of human ESCs of varying sizes indicated that a minimum EB starting size (500 cells/EB) was required for myeloid differentiation to occur in over 90% of EBs and that an intermediate size range (1000 cells/EB) promoted erythroid cell differentiation (47
). The initial size of EBs can also be controlled through the geometric size of microwell or micropattern features in order to spatially define the number of ESCs within individual aggregates (49
). Micropatterned control of ESC colonies can dictate both the size of EBs and the phenotype(s) of the starting cell population used to form EBs, which can affect the differentiation of the cells to particular germ lineages (60
). Recently, microfabricated cell culture inserts compatible with standard multi-well culture plates were reported which significantly enhance the yield of EBs formed using forced aggregation (48
). The size of the resulting EBs can be controlled by the concentration of the cells inoculated into the well and after 24 hours, EBs can be extracted from the microwells with gentle pipetting and transferred to suspension culture. Depending on the dimensions of the microwells, the poly(dimethylsiloxane) inserts contain between 104
microwells per 100 cm2
of surface area - a dramatic increase over the capabilities of round-bottomed 96-well plates (48
In addition to forced aggregation methods, hydrodynamic culture conditions can be used not only to prevent EB agglomeration, but also regulate the size of EBs formed from single cell suspensions (52
). For EBs in horizontal rotary culture and stirred bioreactor culture, an inverse relationship exists between mixing speed and EB size, with decreasing EB size achieved by faster mixing conditions; thus EB size in bulk suspension can be modulated by hydrodynamic mixing conditions (53
). EB size can also be controlled by encapsulating suspensions of individual ESCs or primitive EBs into hydrogel microbeads of controlled volumes. For example, agarose (25
), alginate (61
), and dextran (64
) have all been used successfully to encapsulate ESCs, either as single cells or small clumps of cells, to form EBs within microgels. The diameter of the microgels laden with ESCs can vary greatly from 100 µm agarose beads (25
) to 2.3 mm diameter alginate beads (63
). One problem with increasing microgel size, however, is that encapsulated ESCs may have a tendency to form multiple EBs within individual beads, limiting the ability to accurately control EB size.
Depending on the different culture methods used, the kinetics of EB formation vary dramatically from minutes (forced aggregation) to hours (hydrodynamic mixing) to days (cell encapsulation),. Despite such differences, the consequences of the time scale for EB formation on cell fate and lineage determination has not been directly examined independently of EB size. In addition, although different methods to control initial EB size have been developed, the mechanisms regulating the causal relationship between the size of individual EBs and their propensity to differentiate into different cell phenotypes has yet to be fully elucidated.
2.2. Soluble Factors
Controlling the molecular composition of culture media to direct ESC differentiation has been studied extensively in a variety of systems and the effects of specific soluble factors and signaling pathways on ESC differentiation have been thoroughly discussed previously (65
). Small molecules such as ascorbic acid (68
), retinoic acid (69
) and dexamethasone (70
), as well as larger growth factors such as fibroblast growth factors, bone morphogenic proteins and transforming growth factors (66
), are examples of soluble factors which have been shown to affect ESC differentiation. Presentation of soluble signaling molecules to ESCs in monolayer culture has been the primary method to screen the ability of libraries of chemical compounds and biomolecules to induce ESC differentiation into specific cell types (15
). In lieu of direct co-culture, complex, yet poorly defined media conditioned by secondary cell types has been applied to stem cells in order to direct differentiation (73
). On the other hand, defined soluble media comprised of known amounts of different factors has also been used successfully to generate relatively homogeneous populations of cells, particularly for neural progenitors or neurogenic cell fates (15
In stark contrast to 2D planar culture formats, only the cells on the exterior of 3D EBs are in direct contact with soluble factors present in the culture medium. Soluble factors must diffuse through this multi-layered cell environment and barriers to transport, which likely vary as a function of stages of EB differentiation, contribute to the formation of concentration gradients which comprise the cell microenvironment. Even the diffusion of small molecules (<1000 Da), may have a limited ability to pass through the peripheral cells of EBs (77
). High-powered SEM microscopy analysis of EBs indicates that the surface layer of epithelial-like cells () exhibit tight cell-cell junctions () and cross-sectional analysis of EBs () indicates that EBs tend to form a relatively dense layer of ECM and cells at the periphery of EBs (), compared to the rest of the interior cellular morphology. Therefore, steric barriers to diffusion posed by EB structure make it unlikely that homogenous concentrations of molecules can be attained uniformly throughout the interior of EBs and limit the efficacy of differentiation strategies relying solely on the addition of soluble factors to the culture medium.
2.3. Extracellular Matrix Interactions
The ECM can be a potent mitigator of cell fate decisions by providing a complex assembly of morphogenic cues to stem cells. The ECM is a structural framework of secreted macromolecules consisting primarily of glycosaminoglycans and fibrous proteins which provide mechanical support, adhesive interactions and sequestration of growth factors. Native ECM components direct cell differentiation through integrin-mediated signaling events with adhesive proteins, as well as proteolytic release of affinity-bound growth factors during matrix remodeling (67
). Integrin ligation and growth factor binding to receptors initiate intracellular signaling cascades that ultimately culminate in gene expression changes that modulate cell phenotype (79
The effects of ECM molecules on EB differentiation have largely been examined by seeding ESCs or pre-formed EBs directly within natural ECM hydrogel materials (45
). EBs differentiated in collagen scaffolds consisting of variable amounts of fibronectin and laminin demonstrated that varying the composition of the ECM could differentially direct EB differentiation. EBs in collagen scaffolds with high laminin content adopted a cardiomyocyte phenotype more frequently, whereas EBs were directed towards more epithelial and vascular cell fates in hydrogels with high fibronectin content, and EB cavitation and differentiation appeared to be inhibited in hydrogels with increasing collagen content (80
). ECM signaling peptides can also be incorporated into non-bioactive hydrogels used to encapsulate EBs, such as RGD modified dextran (64
), to examine the effects of ECM on ESC differentiation. In addition to changes in the specific biochemical constituents of the ECM, differences in the elasticity of the ECM may also provide mechanotransductive cues capable of affecting stem cell differentiation (81
Encapsulation of EBs within ECM matrices limits the interactions between ESCs and the ECM to the exterior surface of the ESC aggregates. Therefore, in an attempt to directly manipulate the composition of the ECM within the EB microenvironment, individual matrix molecules like collagen and laminin have been added solubly to suspensions of ESCs during EB formation (17
). Similarly, the addition of soluble complex, tissue-derived matrices, such as Matrigel or Cartigel, to EB culture media has been used to promote the formation of glandular and tubular-like structures or cartilage development, respectively (82
). Although soluble addition of ECM molecules to ESC suspensions may favor incorporation within EBs, soluble ECM molecules alone do not necessarily assemble to form a functional matrix. Self-assembling peptide-based matrices, on the other hand, can rapidly form within developing cell aggregates to form a hydrogel network of nanofibers presenting different signaling epitopes (84
). Utilizing this strategy, neural progenitor cells encapsulated as neurospheres in a self-assembling IKVAV (laminin epitope) amphiphile solution differentiated rapidly into neurons, while astrocyte differentiation was attenuated (85
). Interestingly, the density of the peptide epitope within the cell microenvironment, a material characteristic which can be controlled by matrix formulation conditions, could modulate the differentiation of the cells. Applying a similar principle to EBs, self-assembling matrices could provide a novel route to control the composition and spatial distribution of extracellular signaling motifs present within aggregates of ESCs undergoing differentiation.
2.4. Cell-Cell Interactions
EBs are initially formed via cell-cell adhesive interactions, but intercellular adhesions can also serve an important role in cell signaling throughout EB differentiation. Cell-cell interactions are mediated primarily by cadherins, a family of Ca2+
dependent transmembrane adhesion receptors that play important roles in cell differentiation during embryogenesis (86
). Homophilic cadherin receptor binding triggers intracellular signaling pathways mediated by cytoplasmic catenin proteins, such as β-catenin, which is linked to the Wnt pathway, a potent regulator of cell morphogenesis and differentiation (86
). Undifferentiated ESCs express epithelial-cadherin (E-cadherin), which is the primary molecular mediator of EB formation, but sustained E-cadherin expression can also be responsible for the agglomeration of EBs at later stages of differentiation (25
). Inhibition of E-cadherin mediated adhesion, either by the use of E-cadherin binding antibodies or E-cadherin null ESCs, prevents normal EB formation and subsequent differentiation (25
). Differential cadherin expression, associated with different cell phenotypes, is temporally regulated during the course of EB differentiation and can directly influence cell fate specification. For example, ESCs constitutively expressing E-cadherin are prone to more epithelial differentiation, while ESCs constitutively expressing N-cadherin differentiate more readily into cartilage and neuroepithelium (27
). Although it has not been systematically investigated, the use of cadherin signaling to control EB differentiation either through integration of a genetically modified cell line over-expressing a particular cadherin or through the presentation of cadherins on biomaterial surfaces integrated within EBs to mimic cell-cell interactions is a promising area for control of EB differentiation.
Strategies to control other types of cell-cell interactions, including transmembrane receptors and ligands not anchored to the cytoskeleton, have also been explored in stem cells. For example, the Notch pathway is involved in a variety of cell fate decision processes through development and adult tissue morphogenesis (87
). ESCs can express multiple Notch receptors and Notch signaling has been implicated both in stem cell self-renewal and differentiation towards different phenotypes, such as neuronal cells (89
). In general, Notch signaling requires immobilized ligand presentation from a surface or cell membrane in order to achieve optimal bioactivity (92
). Jagged-1, a Notch ligand, immobilized to polystyrene or polyHEMA surfaces promoted early and late stage differentiation of cultured epithelial stem cells (93
). Comparable methods of presenting Notch ligands to cells uniformly within EBs would require that engineered biomaterials be integrated directly within the interior of the ESC aggregate.