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

FAGUNDEZ, CAROL BETIANA; LORESI, MÓNICA ALEJANDRA; DELCOURT, STELLA MARIS; ARGIBAY, PABLO FRANCISCO

Methodological Advances in the Culture, Manipulation and Utilization of Embryonic Stem Cells for Basic and Practical Applications

Intech

Año: 2011; p. 25 - 44

The intention of this chapter is to provide an overview of mouse embryonic stem cells, presenting useful and practical advices to those working in this field, including some personal experience of the authors in this topic.   A stem cell has the capacity to self-renew for long-term periods without losing its ability to differentiate itself to more specialized cell types (pluripotency). Through asymmetric divisions, it generates, on the one hand, a cell which preserves the stemness state (self-renewal and pluripotency) and, on the other hand, a differentiated cell which continues dividing itself symmetrically (Rippon and Bishop, 2004). Mouse embryonic stem cells (mESCs) are generally obtained from the ICM of preimplantation blastocyst-stage embryos. Although these cells display a relatively short lifespan in vivo, they can be cultured indefinitely in an undifferentiated state if anti-differentiation factors like leukaemia inhibitory factor (LIF) are added to the medium, or when co-cultured with a feeder layer of mitotically inactivated murine embryonic fibroblasts (MEFs) (Baharvand and Matthaei, 2003, 2004; Rippon and Bishop, 2004). In 1981, Evans and Kaufman successfully isolated mESCs culturing whole blastocysts on a MEFs feeder layer obtaining what they later called EK cells. These cells showed a stable karyotype and were able to produce teratocarcinomas when injected into immunodeficient mice, as well as to form cystic embryoid bodies (EBs) when grown in suspension (Evans and Kaufman, 1981). Some time later, Martin et al. could isolate pluripotent cells derived from the ICM of blastocysts culturing them in presence of embryonal carcinoma cells (ECC) conditioned medium. Martin named these cells “embryonic stem cells”, to indicate their origin from embryos and to distinguish them from the ECC, coming from teratocarcinomas (Martin, 1981). Embryonic stem cells share many properties with the pluripotent cells from the ICM, including expression of pluripotent markers like Oct4, Nanog and Sox2. These three transcription factors are key regulators in a network of target genes which collaborate in maintenance of the main properties of ESCs, self-renewal and pluripotency (Boyer et al., 2006; Wobus and Boheler, 2005). Presence of cell surface markers as SSEA-1 are also displayed by these cells, as well as high activity of certain enzymes, like alkaline phosphatase (ALP) and telomerase, the last one being related to immortality of these cells (Solter and Knowles, 1978; Wobus and Boheler, 2005). Mouse ESCs show unique cell morphology, with a high nucleus/cytoplasm ratio and colonies growing like cummulus, in a multilayered manner. Derivation of mESCs is strongly mouse strain-dependent (Baharvand and Matthaei, 2004; Bryja et al., 2006; Kawase et al., 1994). In our laboratory, we derived mESCs from two strains, Balb-c and C57BL/6, with a higher efficiency obtained by the first one, supporting this statement. The ability of mESCs to give rise to derivatives of the three germ layers needs to be corroborated in vitro inducing formation of EBs, and in vivo by formation of teratomas and chimeras. Mouse embryonic stem cells have become a powerful tool for biomedical research and have played an important part in elucidating many of the signal transduction pathways that regulate progression through the cell cycle. By means of transgenic techniques, they have also proved their potential for modelling human diseases, providing an opportunity to unravel biochemical, genetic and/or physiological causes of these pathologies.