Phenotypic characteristics of feline adipose-derived stem cells affected by cell passage number.

In this study we evaluated the influence of passage number on the phenotypic characteristics of feline adipose-derived stem cells (ASCs) in order to develop a broader understanding of their dynamics. Feline ASCs were individually isolated from five domestic cats and subjected to proliferative culture at passage (P) 2, 6 and 10. The cells at each sub-passage were examined in regard to their phenotypic properties associated with multipotent mesenchymal stem cells (MSCs), such as morphology, proliferation kinetics, self-renewal, and expression of MSCs-specific surface markers. The differentiation capacity into adipocytes and osteoblasts was also identified. Feline ASCs appeared with a fibroblast-like morphology with minimal alteration through P10. The rate of cell proliferation gradually decreased, while cell doubling time gradually increased with each passage. A significant decrease in CFU-F efficiency was observed with increasing cell passage number. The ASC population uniformly expressed their characteristic markers CD44 and CD90, but did not express the hematopoietic marker CD45. However, MSC markers gradually decreased in the later passage stages. Feline ASCs were capable of undergoing both adipogenesis and osteogenesis at P2. These findings suggested that the phenotypic characteristics of feline ASCs could be affected by long-term passages, which is potentially very important in regard to their therapeutic application.

Fivefold increase in derivation rates of mouse embryonic stem cells after supplementation of the media with multiple factors.

The objective of this study was to determine the ability of multiple-factor supplementation to augment derivation of mouse embryonic stem (mES) cells. Three factors, leukemia inhibitory factor (LIF), Parke-Davis 98059 (PD98059), and 6-bromoindirubin-3'-oxime (BIO), were added as supplements (individually or in a combination of all three) at two consecutive stages of culture; that is, from the start of blastocyst culture to the outgrowth stage, and from putting disaggregated outgrowth into culture medium to generation of primary mES colonies, respectively. The main outcome measure was the percentage of derivable mES cell lines, based on the number of blastocysts initially cultured. Three experiments demonstrated the following: (1) For the addition of individual single factor, only LIF yielded mES cell lines (6.2%), whereas a combination of all three factors resulted in the greatest number of mES cell lines (31.3%). (2) The advantages of a combination of multiple factors (LIF+PD98059+BIO) were manifested only when they were used during the first stage of the culture and not during the second stage (31.6% vs. 6.2%, respectively). (3) The quality of the inner cell mass (ICM) outgrowth obtained from first-stage culture was studied. After alkaline phosphatase and Oct-4 staining, which documented pluripotency of the embryonic stem cells, outgrowths cultured in multiple factors (LIF+PD98059+BIO) stained much stronger and in higher proportions than did those obtained after supplementation only with LIF (80% vs. 30%, respectively).