A simple approach for mouse embryonic stem cells isolation and differentiation inducing embryoid body formation

Abstract

Stem cells were derived from hatched blastocyst-stage mouse embryos of the C57BL/6 strain employing a knockout serum replacement instead of the traditional fetal calf serum, thereby avoiding the use of immunosurgery. Although fetal calf serum was not good for isolation of stem cells, a combination of this serum plus knockout serum increased the expansion rate of the cell culture. The derived cells were capable of maintaining an undifferentiated state during several passages, as demonstrated by the presence of alkaline phosphatase activity, stage-specific embryonic antigen 1 (SSEA-1), and octamer binding protein 4 (Oct-4). Suspension culture in bacteriological dishes gave better results than the hanging drop method for differentiation by means of embryoid body formation. Mouse embryonic stem cells showed spontaneous differentiation into derivatives of the 3 germ layers in culture media supplemented with fetal calf serum but not with knockout serum.

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

“…When staining of mESCs with the cell surface marker SSEA-1 was carried out in our laboratory, the extensions projected to the MEFs mentioned above were observed, supporting this information (Fig. 2E) (Fagundez et al, 2009). …”

“…Reichert-like basement membrane deposits are often seen during the first days of EB culture and its presence can be identified by Periodic Acid Shiff (PAS) reaction (Fig. 4A,B) (Fagundez et al, 2009;Zhou et al, 2005). There are different types of culture media that can be employed to induce formation of EBs and results vary according to the medium chosen and whether they are supplemented either with FBS or Ko-SR. As mentioned earlier, FBS contains pro-differentiation factors which make it a useful tool in differentiation protocols (Fagundez et al, 2009).…”

“…Some authors have attempted supplementation of ES medium with a FBS-KoSR mixture in different ratios to improve the isolation and the subsequent establishment of ESCs (Lee et al, 2006;Tanimoto et al, 2008). However, culture in ES medium without complete or partial FBS replacement do not always support ESCs derivation, but could be beneficial to increase cell growth rate of the colonies already established, while preserving the stemness state (Fagundez et al, 2009;Lee et al, 2006). The use of KoSR could be useful to easily isolate mESCs from cultures of whole blastocyst embryos, since it prevents development of trophoblastic cells which tend to colonize the culture, avoiding also the application of immunosurgery, a complex technique involving the use of cytotoxic antibodies, that is not always efficient at the moment of eliminating all the trophoblastic cell population of the embryo (Fagundez et al, 2009;Gardner, 1985).…”

“…The cells do not attach to the plastic surface of the dish; instead they stick to each other forming aggregates that will give rise to EBs (Kurosawa, 2007). Another way to form EBs employing these plates is to culture the whole colonies of mESCs during varying periods of time (Fagundez et al, 2009). Unfortunately, both of these methods produce heterogeneous EBs with distinct degrees of differentiation.…”

“…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.…”