284 CHARACTERIZATION OF GOAT MESENCHYMAL STEM CELLS DERIVED FROM BONE MARROW AND ADIPOSE TISSUE

Abstract

Gene modification of cells in vitro followed by somatic cell nuclear transfer (SCNT) currently offers the best route for creating genetically modified livestock species. However, low cloning efficiencies in differentiated somatic cells have been attributed to the possibility of improper nuclear reprogramming. Adult stem cells may have greater developmental potential and better nuclear reprogramming potential following cloning. There is considerable interest in using goats as models for genetically engineering dairy animals and for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells (MSC) are adult stem cells that that have been isolated and characterised from various species, but are poorly characterised in goats. Three MSC lines were isolated from bone marrow (9004 BM-MSC, 9003 BM-MSC) and adipose tissue (9003 A-MSC) of neonatal goats. In this study, these MSC lines were characterised to verify MSC-specific characteristics and assess their amenability to genetic modification in vitro. Passage 5 cells were evaluated for capacity to differentiate into osteogenic, adipogenic, and chondrogenic lineages, as well as for colony-forming efficiency after 10 days of culture from low-density plating. Expression of MSC-specific positive cell surface markers CD90, CD73, and CD105, as well as pluripotency markers Nanog, Oct-4, and Sox-2, was examined by RT-PCR. Oct-4 protein localization was examined by immunofluorescence. The MSC were also assessed for their potential for gene modification by nucleofection with circular and linearized plasmids expressing green fluorescent protein (GFP) and neomycin resistance. Differences between cell lines were statistically analysed using ANOVA. The 9003 BM-MSC cells were also utilised for SCNT. All 3 MSC lines showed a normal karyotype. The MSC lines were capable of undergoing osteogenic, adipogenic, and chondrogenic differentiation, with observed differences in capacities between the BM-MSC and A-MSC lines, as shown by staining with Alizarin Red S, Oil Red O, and Alcian Blue. Expression of CD90, CD73, CD105, Nanog, Oct-4, and Sox-2 was detected, and Oct-4 was localised in the cytoplasm. There were significant differences in clonability between the cell lines, with 9004 BM-MSC showing the highest colony-forming efficiency (61% ± 5.4; P < 0.05). There were no significant differences in the percentage of GFP-positive cells from transfections done with the circular plasmid, but 9003 BM-MSC yielded a significantly lower number of integrant colonies per 500 000 cells transfected with the linear plasmid and G418 selection (12.75 ± 3.24; P < 0.05). Somatic cell nuclear transfer was able to reprogram 9003 BM-MSC and produce pregnancies. One hundred forty-four embryos were reconstructed, 101 embryos were transferred into 8 recipients, and the resulting pregnancy rate was 73%. Our findings provide characterisation information on goat MSC, and show that significant differences can exist between MSC isolated from different tissues and from within the same tissue.