Isolation, Characterization, Genetic Modification and Nuclear Reprogramming of Porcine Mesenchymal Stem Cells

BOSCH, P.; PRATT, S. L.; STICE, S. L.

BIOLOGY OF REPRODUCTION

Society for the Study of Reproduction

Año: 2006 vol. 74 p. 46 - 57

0006-3363

Bone marrow mesenchymal stem cells (MSCs) are adult pluripotent cells considered an important resource for human cell-based therapies. Understanding MSC clinical potential may require their use in preclinical large animal models such as pigs. The objectives of this study were: 1) to establish porcine MSC (pMSC) cultures; 2) to optimize in vitro pMSC culture conditions 3) to investigate whether pMSCs are amenable to genetic manipulation; and 4) to determine pMSC reprogramming potential using somatic cell nuclear transfer (SCNT). pMSCs isolated from bone marrow grew, attached to plastic with a fibroblast-like morphology, and expressed the mesenchymal surface marker THY1 but not the hematopoietic marker ITGAM. Furthermore, pMSCs underwent lipogenic, chondrogenic and osteogenic differentiation when exposed to specific inducing conditions. Porcine MSCs grew well in a variety of media and proliferative capacity was enhanced by culture under low oxygen atmosphere. Transient transduction of pMSCs and isogenic skin fibroblasts (SFs) with a human adenovirus carrying the gene for green fluorescent protein (GFP) (Ad5-F35eGFP) resulted in more pMSCs expressing GFP compared with SFs. Cell lines with stable genetic modifications and extended expression of transgene were obtained when pMSCs were transfected with a plasmid containing the GFP gene. Infection of pMSC and SF cell lines by an adeno-associated virus resulted in ~12% transgenic cells, which formed transgenic clonal lines after propagation as single cells. pMSCs can be expanded in vitro and used as nuclear donors to produce SCNT embryos. Thus, pMSCs are an attractive cell type for large animal autologous and allogenic cell therapy models and SCNT transgenesis.