The isolation and characterization of bovine adult derived adipose stem cells for the use in nuclear transfer

Since the cloning of Dolly there has been little change in the efficiency of nuclear transfer (NT). Research is beginning to investigate the characteristics of donor cells. Adiposetissue is an abundant source of adult-derived cells that have displayed “stemness” in-vitro(Gimble et al., 2003). The overall goal of this research was to define the in-vitro characteristicsof bovine adipose-derived adult stem cells (ADAS) for the use in NT. Isolation methods weredetermined by a 3 x 3 factorial design. 1 g of subcutaneous fat was collected and subjected to0.10%, 0.25% or 0.50% collagenase type I solution for 1, 2 and 3 h. Nucleated cells werecounted using heochst stain. There was no significant difference (P\u3e0.05) in number of nucleated cells released during the incubation period or collagenase concentrations. Viable cells were determined by those that remained adherent 24 h post plating. Incubation in 0.25% collagenase for 2 h had the consistently highest percentage of viable cells (45%). The lifespan and growth characteristics were determined by in a 2 x 2 factorial experiment of DMEM or DMEM:F12 supplemented or not supplemented with growth factors. DMEM with growth factors supplementation was significantly shorter lifespan (P\u3e0.05) than DMEM:F12. The averagelifespan was ~30 population doublings (PDs), with 1 cell cycle every two days until passage 8(P8). Two bovine ADAS cell lines were differentiated into adipocytes, chondrocytes and osteoblasts at middle and late passages along side of adult derived skin fibroblasts. Differentiation was confirmed by histological staining resulting in early passage ADAS cells staining more intensely compared to late passage ADAS cells and skin fibroblasts. Global levels of DNA methylation and histone acetylation were analyzed from P1 to P6 in ADAS and skin fibroblasts from three animals. There was no significant difference (P\u3e0.05) between cell types for DNA methylation or histone acetylation. The percentage of cleaved and developing blastocyst embryos from the ADAS cells (62% and 8%) and skin fibroblasts cells (42% and 8%)were not different (P\u3e0.05). Interspecies nuclear transfer utilized eland ADAS cells into enucleated bovine oocytes. A total of 3 interspecies embryos (1%) developed to blastocyst

Isolation and characterization of porcine adipose tissue-derived adult stem cells

Background: Stem cell characteristics such as self-renewal, differentiation and expression of CD34 and CD44 stem cell markers have not been identified in porcine adipose tissue-derived adult stem (ADAS) cells. The objective of this study was to develop a protocol for the isolation and culture of porcine adipose tissue-derived cells and to determine stem cell-like characteristics. Methods: Primary cultures were established and cell cultures were maintained. Cloning capacity was determined using a ring cloning procedure. Primary cultures and clones were differentiated and stained for multiple differentiated phenotypes. CD34 and CD44 messenger ribonucleic acid (mRNA) was isolated and reverse transcriptase polymerase chain reaction was used to compare expression profiles. Results: An average of 2,700,000 nucleated cells/ml was isolated; 26% were adherent, and cells completed a cell cycle approximately every 3.3 days. Ring cloning identified 19 colonies. Primary cultures and clones were determined to differentiate along osteogenic, adipogenic and chondrogenic tissue lineages. The mRNA expression profiles showed CD34 expression was higher for undifferentiated ADAS cells versus differentiated cell types and the CD34 expression level was lower than that of CD44 among differentiated cells. Conclusion: Improved culture conditions and defined cellular characteristics of these porcine ADAS cells have been identified. Porcine ADAS can self-renew, can differentiate into multiple tissue lineages and they express CD34. Copyright © 2008 S. Karger AG