Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos . The abilities of ES cells to undergo indefinite self-renewal in vitro and to produce derivative lineages of all three embryonic germ layers in vitro and in vivo make them highly prized in both clinical and research settings . ES, or ES-like, cells have thus far been derived from a number of mammalian species, including the mouse , rat , bovine , sheep , pig , rhesus macaque , crab-eating macaque , marmoset  and human .
Apodemus sylvaticus is a common rodent species found throughout Europe. A. sylvaticus has a gross appearance similar to that of the laboratory mouse. The rearing conditions are also quite similar to those of the mouse. However, the superficial resemblance between A. sylvaticus and the laboratory mouse belies the rather deep evolutionary divide separating these two species. The combination of these properties--that is, the similar rearing conditions and large evolutionary divergence--makes A. sylvaticus highly attractive as a potential model organism that could perhaps complement the mouse in many studies. Unlike the mouse, however, there is a dearth of knowledge and reagents related to A. sylvaticus. One major step in filling this gap is the generation of ES cells for this species. Recently, we reported the successful establishment of an ES cell line from A. sylvaticus
, named AS-ES1 cells. This cell line has proliferated continuously for over 6 months with a normal karyotype. It expresses a variety of markers associated with the undifferentiated state and has the ability to produce lineages of all three germ layers in vitro and in vivo. However, there are some characteristic differences between AS-ES1 and mouse ES cells. For example, AS-ES1 cells do not express stage specific embryonic antigen-1 (SSEA-1), whereas mouse ES cells do. Furthermore, the AS-ES1 cell line proliferates faster than specific mouse ES cell lines. Therefore, as a new species of ES cell line, the basic characteristics of AS-ES1 cells need to be studied further, including specific lineage differentiation.
Mouse ES cells were first established in 1981. Since then, many studies have been carried out regarding the three lineages differentiation of mouse ES cells in vitro. For mesodermal differentiation of mouse ES cells in vitro, different research groups have generated a variety of cell types, such as adipocytes [13, 14], osteoblasts [15–19], chondrocytes [20–22] and cardiomyocytes [23, 24], among others. Through this research, some pivotal agents that play an important role in the process of mesodermal differentiation of mouse ES cells have been discovered. However, it was not known whether those agents and differentiation methods could work with AS-ES1 cells.
Herein we report that AS-ES1 cells treated with retinoic acid (RA) or 5-azacytidine (5-AZA) at the embryoid body (EB) stage, with the addition of various specific factors and reagents to the medium during EB attachment, generated multiple mesodermal lineages in vitro, including adipocytes, osteoblasts, chondrocytes and cardiomyocytes.