The efficient formation of highly organized, cystic EBs is significant for a number of reasons, including improved in vitro
models of embryogenesis and biomanufacturing of differentiated cell types for regenerative medicine and in vitro
diagnostic applications. Currently, directed differentiation strategies using EBs are limited in their ability to produce a homogeneous differentiated cell population, due largely to the fact that EBs consist of a population of cells exposed to a diverse set of instructive cues within the microenvironment [33
]. The variability in the interactions of different cells within an EB with soluble morphogens, extracellular matrix, and neighboring cells leads to significant heterogeneity in cell differentiation microscopically within an individual EB and macroscopically among a population of EBs [36
]. However, by specifying EB differentiation to two cell phenotypes initially – visceral endoderm and epiblast – it may be possible to subsequently direct cells more efficiently to specific lineages by reducing endogenous signaling, thereby allowing defined media components to more potently dictate cell fate decisions. Moreover, while untreated EBs contain a matrix-dense outer layer that appears to hinder diffusion of soluble media components, RA MS EBs contain an outer cell layer densely coated with microvilli. The appearance of microvilli strongly suggests that the outer cells may be proficient in the absorption of nutrients and soluble signaling molecules from the media, allowing such molecules to reach the interior epiblast cells more efficiently and thereby enhance subsequent directed differentiation.
The striking similarity of RA MS EBs to E6.75 embryos has significant implications for in vitro
studies of embryogenesis. EBs have been utilized to study the roles of specific genes in development, particularly for genes in which knockout models in vivo
are unavailable due to embryonic lethality [37
]. However, EBs formed using conventional methods may not provide an accurate representation of the roles of such genes in later stages of embryogenesis, since EBs commonly differentiate in a heterogeneous, disorganized manner within a fairly short period of time (<7 days). The use of RA MS EBs may provide greater insight into developmental processes due to their close phenotypic resemblance to early streak (E6.75) embryos [22
]. Additionally, the roles of specific secreted molecules during development can be studied in the well controlled, in vitro
environment created using RA MS EBs, which may lead to a better understanding of early differentiation events, such as axis formation, specification of epiblast cells, and germ layer formation.
RA treatment of ESCs most commonly promotes neurogenesis [39
]; however, enhanced cardiogenesis [45
] and pancreatic beta-like cell formation [46
] in response to RA have also been reported, indicating this retinol-derived morphogen is a potent inducer of differentiation to various cell lineages. Similar to the effects of RA in vivo
], the concentration of RA and the timing of its delivery appear to be critical in dictating cell differentiation in vitro
]. We report here that delivery of RA directly within the EB interior via microspheres maintains the pluripotency of ESCs through the formation of a highly organized OCT4+ epiblast, suggesting that the mode of presentation of RA (media supplement vs. microsphere-mediated) is a key determinant of cellular response, as soluble RA treatment at a wide range of concentrations [10 nM–10 μM] was unable to efficiently produce comparable cystic EBs. These discrepancies may be attributed to the fact that soluble factor delivery only affects cells at the exterior surface of EBs and prevents the remaining interior cells from being directly exposed to exogenous biochemical stimuli, such as RA. Interestingly, ESCs that constitutively express Nodal
, a member of the TGF-β superfamily, form cystic spheroids with similar characteristics as RA MS EBs, whereas addition of recombinant Nodal to the culture media did not elicit a similar effect [49
]. This suggests that 1) diffusion of Nodal into EBs is restricted and results in distinct responses between soluble delivery and constitutive local expression, and 2) the RA and TGF-β signaling pathways may share common elements [50
], resulting in similar outcomes for local RA delivery and Nodal over-expression. Differentiation of EBs in media conditioned by HepG2 cells, a hepatocarcinoma-derived endodermal cell line, induces formation of uniform cystic EBs that lack an outer endoderm cell layer [30
]. This suggests that in the absences of an epithelial exterior, morphogens contained within the conditioned media may more efficiently diffuse into EBs and direct differentiation. Thus, the diffusional limitations posed by EBs may be an underappreciated facet of ESC biology that may lead to an incomplete understanding of the roles of specific molecules in ESC differentiation [15
Previous studies have reported the incorporation of PLGA microspheres containing growth factors into spheroids of fetal brain cells [16
] as well as human EBs [53
] in order to manipulate the biochemistry of 3D spheroid microenvironments. PLGA microspheres containing nerve growth factor (NGF) were assembled with fetal rat brain cells to form programmable neo-tissues and transplanted into adult rats. The controlled-release microspheres were able to support transplanted cell survival, but specific effects on cell differentiation were not described or compared directly to soluble delivery methods. Recently, microspheres containing angiogenic growth factors (vascular endothelial, placenta and basic fibroblast growth factors) were incorporated into EBs using forced aggregation, and expression of vascular cell markers was assessed after 10 days of differentiation. However, compared to soluble growth factor treatment, this approach did not improve vascular differentiation of human ESCs, likely due to the lack of release of growth factors after 24 h and the low overall growth factor availability. In contrast, small hydrophobic molecules (such as RA) are readily incorporated into PLGA microspheres using a single emulsion and release is sustained over the course of days–weeks, allowing sufficient morphogen availability within EBs to elicit significant differences in ESC differentiation compared to soluble delivery methods.