394 CHARACTERIZATION OF BOVINE ADULT-DERIVED ADIPOSE STEM CELLS FOR USE IN NUCLEAR TRANSFERA. A. Picou A , J. Wilson A , B. Dresser B , G. T. Gentry A , R. A. Godke A and K. R. Bondioli A
A Embryo Biotechnology Laboratory, School of Animal Sciences, Louisiana State University Agricultural Center, Baton Rouge, LA, USA;
B Audubon Nature Institute Center for Research of Endangered Species, New Orleans, LA, USA
Reproduction, Fertility and Development 22(1) 353-354 http://dx.doi.org/10.1071/RDv22n1Ab394
Published: 8 December 2009
Adipose tissue is an abundant source of adult-derived cells that have displayed multipotent properties in vitro. The goal of this research was to study the characteristics of bovine adipose-derived adult stem (ADAS) cells to determine the feasibility for use in NT. Adipose tissue was isolated from the brisket of adult cattle postmortem. Cells were isolated by incubation for 2 h with 0.25% collagenase solution, separation of stromal cells by centrifugation, and selection by adherence to plastic. The lifespan and growth characteristics for culture conditions were determined by a 2 × 2 factorial with DMEM or DMEM:F12 and with or without growth factor (GF) supplementation.A two-way ANOVA, followed by multiple pair-wise comparisons using Tukey’s test when applicable, was used to detect differences in population doublings (PD) until senescence for media treatments and GF supplementation. Dulbecco’s modified Eagle’s medium with GF supported significantly less (PD) (P > 0.05) than DMEM : F12. The average lifespan was approximately 30 PD, with a cell length of 48 h until passage 8 (P8). As cells approached replicative senescence, the cell cycle length was inconsistent. Two ADAS and one adult-derived skin fibroblast cell lines from different animals were subjected to differentiation conditions for adipocytes, chondrocytes, and osteoblasts at P2, P6, and P11. Differentiation was confirmed by histological staining. Passage 2 ADAS cells differentiated more efficiently than did P6, P11, or skin fibroblasts. Global levels of DNA methylation and histone acetylation were analyzed from P1 to P6 in 3 sets of cell lines consisting of ADAS and skin cells from the same animals by immune staining and flow cytometry. There was no significant difference (P > 0.05) between cell types by one-way ANOVA. Nuclear transfer was performed using ADAS cells as donor cells and commercially supplied oocytes. Mature, enucleated oocytes were reconstructed with either adult skin fibroblasts or ADAS cells. The percentage of cleaved and blastocysts from ADAS cells (62% and 8%, n = 163) and skin fibroblasts cells (42% and 8%, n = 170) were not different (P > 0.05) by chi-square analysis. Interspecies NT was attempted with eland (Taurotragus oryx) ADAS cells and enucleated bovine oocytes. Two groups of enucleated oocytes were reconstructed with bovine (n = 234) and eland (n = 290) ADAS cells. There was no significant difference between the number of cleaved embryos (38% and 39%) or blastocysts formed by chi-square analysis. A total of 3 interspecies embryos (1%) and 5 bovine embryos (14%) developed to blastocysts. Bovine ADAS cells are not more efficient than bovine adult-derived skin fibroblasts as donor cells, but they do represent a viable option for use in NT because of their higher in vitro development. Eland ADAS cells resulted in development to the blastocyst stage after interspecies NT.