Culture and differentiation of large animals stem cells.

The development of stem cell technologies in large animals may serve both as a tool for engineering the genome and as a model of cell transplantation for regenerative medicine. Bovine, sheep, pig and horse have been used for the derivation and culture of both embryonic and mesenchymal stem cells. The derivation of embryonic stem cells relies on the large supply of fertilised in vitro produced pre-implantation embryos. Derivation from parthenogenetic, nuclear transfer and interspecies nuclear transfer embryos have also been described. Most of the cell lines generated from large animals embryos have only some of the features of stemness that are considered essential for mouse or human embryonic stem cells, and usually undergo spontaneous differentiation after a short period in culture. Under culture conditions that favor neural differentiation we have derived neural precursor cell lines from bovine and ovine embryos. In particular we obtained bovine neural precursor cell lines from nuclear transfer embryos with the same efficiency of normal embryos. These neural cell lines under appropriate condition can be induced to differentiate into neural crest derivatives including smooth muscle and cartilage. Although the derivation of intergenera nuclear transfer embryonic stem cell has been described, the development of such embryos in large animals is severely compromised in our experience and it is therefore been impossible to derive cell lines from them. Mesenchymal stem cells are easily collected from bone marrow and adipose tissue from the four species mentioned. They have a finite life span (from 40 to 70 population doublings) and retain the ability to differentiate into derivatives of the mesenchyme including cartilage, bone and fat. These cells have been successfully marked with eGFP by both lentivirus and electroporation.