In Vitro Differentiation of Mouse Embryonic Stem Cells: Enrichment of Endodermal Cells in the Embryoid Body

Abstract

Embryonic stem (ES) cells have the potential to differentiate into all three germ layers, providing new perspectives not only for embryonic development but also for the application in cell replacement therapies. Even though the formation of an embryoid body (EB) in a suspension culture has been the most popular method to differentiate ES cells into a wide range of cells, not much is known about the characteristics of EB cells. To this end, we investigated the process of EB formation in the suspension culture of ES cells at weekly intervals for up to 6 weeks. We observed that the central apoptotic area is most active in the first week of EB formation and that the cell adhesion molecules, except β-catenin, are highly expressed throughout the examination period. The sequential expression of endodermal genes in EBs during the 6-week culture correlated closely with that of normal embryo development. The outer surface of EBs stained positive for α-fetoprotein and GATA-4. When isolated from the 2-week-old EB by trypsin treatment, these endodermal lineage cells matured in vitro into hepatocytes upon stimulation with various hepatotrophic factors. In conclusion, our results demonstrate that endodermal cells can be retrieved from EBs and matured into specific cell types, opening new therapeutic usage of these in vitro differentiated cells in the cell replacement therapy of various diseases. Stem Cells 2005;23:817-827

Controlled embryoid body formation via surface modification and avidin–biotin cross-linking

“…The core composed necrotic ES cells and the surface composed proliferating ES cells. The surface was denser than the core, supporting the idea that the EB surface may have acted as a thick protective barrier that inadvertently restricted nutrient and gaseous permeation (Choi et al 2005;Sachlos and Auguste 2008). The outer and inner shells combine to represent the diffusion gradient of nutrient and gaseous exchange depicting transition between ES cell states at the surface and core (Jiang et al 2005).…”

Developmental potential of Gcn5−/− embryonic stem cells in vivo and in vitro

“…For example, T(Bra), an early marker for mesoderm, was not highly expressed until D6 in wild-type EBs, but robust expression of T(Bra) was observed by D3 in Gcn5 Ϫ/Ϫ EBs and continued until D9. Expression of another mesodermal marker, Flk1 (Elefanty et al, 1997;Choi et al, 2005), was slightly higher at early times (D3 and D6) but was terminated prematurely in Gcn5 Ϫ/Ϫ EBs by D9. Expression of a third mesodermal marker, Goosecoid (Gsc; Fig.…”

Mouse Embryonic Stem Cells Basics from a Fertilized Zygote to These Promising Pluripotent Stem Cells

“…In our laboratory we were not able to form EBs with HD technique, as cells were unable to group and failed to produce three-dimensional aggregates. Although generation of EBs in suspension culture employing whole ESCs colonies has some disadvantages, it seems possible that the proximity established among cells when growing together could be an important factor favouring the differentiation process, since cell to cell contact plays a major role in the early stages of embryonic development (Choi et al, 2005;Fagundez et al, 2009). Semisolid suspension culture.…”

“…Mutual cell to cell interactions are important for the differentiation of stem cells into the cell types derived from the three germ layers. Cell-cell contact and apoptosis are very important during the early developmental stages and are expected to be found during the formation of embryoid bodies (Choi et al, 2005;Doetschman et al, 1985). Apoptosis is a natural process of development and several studies have revealed that apoptosis-related genes are involved in the differentiation of ESCs to EBs (Choi et al, 2005).…”

“…Cell-cell contact and apoptosis are very important during the early developmental stages and are expected to be found during the formation of embryoid bodies (Choi et al, 2005;Doetschman et al, 1985). Apoptosis is a natural process of development and several studies have revealed that apoptosis-related genes are involved in the differentiation of ESCs to EBs (Choi et al, 2005). During EB formation, multiple foci of cell death appear to finally merge into a single central cavity surrounded by a monolayer of primitive ectoderm cells (Fig.…”