Establishment, differentiation, electroporation, viral transduction, and nuclear transfer of bovine and porcine mesenchymal stem cells.
Mesenchymal stem cells (MSCs) reside in the bone marrow and have the potential for multilineage differentiation, into bone, cartilage, and fat, for example. In this study, bovine and porcine MSCs were isolated, cultured to determine their replication ability, and differentiated with osteogenic medium and 5-azacytine. Both bovine and porcine undifferentiated MSCs were electroporated and virally transduced to test the efficiency of genetic modification and the maintainance of differentiation ability thereafter. Nuclear transfer experiments were carried out with bovine and porcine MSCs, both at the undifferentiated state and following differentiation. Our results indicate that bovine and porcine MSCs have limited lifespans in vitro--approximately 50 population doublings. They can be efficiently differentiated and characterized along the osteogenic lineage by morphology, alkaline phosphatase, Von Kossa, oil red stainings, and RT-PCR. Electroporation and selection induce high levels of EGFP expression in porcine but not in bovine MSCs. Following genetic modification, MSCs retain their pluridifferentiation ability as parental cells. Cloned embryos derived from bovine and porcine undifferentiated MSCs and their derivatives along the osteogenic lineage give rise to consistently high preimplantation development comparable to adult fibroblasts.
Colleoni
S
S
Laboratorio di Tecnologie della Riproduzione, CIZ srl, Istituto Sperimentale Italiano Lazzaro Spallanzani, Cremona, Italy.
Donofrio
G
G
Lagutina
I
I
Duchi
R
R
Galli
C
C
Lazzari
G
G
D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division; D019070:Cell Lineage; D002467:Cell Nucleus / Q000502:physiology; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods; D003596:Cytosine / Q000031:analogs & derivatives / Q000494:pharmacology; D018274:Electroporation / Q000379:methods; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology*; D053652:Nuclear Transfer Techniques; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D013552:Swine; D014161:Transduction, Genetic / Q000379:methods
C039854:5-azacytosine; D003596:Cytosine