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).

Fundamental cryobiology of mammalian oocytes and ovarian tissue.

Embryo cryopreservation is a widely used and relatively well-established procedure. By contrast, ovarian tissue and unfertilized oocytes are only rarely cryopreserved, even though for germ line storage these often would be preferable to embryo cryopreservation. There are many reasons for this discrepancy. Unfertilized mature (MII) stage oocytes are more difficult to cryopreserve than cleavage stage embryos of the same species. Many factors contribute to this including the oocyte's surface to volume ratio, single membrane, temperature-sensitive metaphase spindle and zona, and its susceptibility to parthenogenetic activation and chill-injury. A completely different set of problems applies to primordial follicles. Oocytes in primordial follicles are very small and tolerate cryopreservation by slow cooling very well. The problem lies in the difficulty in producing mature oocytes from these primordial follicles. Better and/or more convenient cryopreservation procedures for both oocytes and ovarian tissue are being developed. This paper describes some of the advances in this area and outlines the relative merits and limitations of several currently available egg and ovarian tissue cryopreservation procedures.

Lack of effect of oral contraceptive use on the pharmacokinetics of quinine.

The pharmacokinetics of quinine after a single 600 mg oral dose of quinine sulphate were studied in seven female subjects who used oral contraceptives and in seven age-matched female controls who did not. There were no significant differences (P greater than 0.05) in the maximum plasma concentration (Cmax) and the time of peak concentration (tmax) between the subjects who used oral contraceptives (Cmax = 5.3 +/- 1.0 (s.d.) mg l-1; tmax = 1.4 +/- 0.7 h) and the control subjects (Cmax = 5.6 +/- 0.9 mg l-1; tmax = 2.1 +/- 0.9 h). The mean elimination half-life of quinine in the oral contraceptives user group (12.5 +/- 1.9 h) was similar (P greater than 0.05) to that in the control group (11.8 +/- 2.7 h). The oral clearance of quinine in the oral contraceptive user group was 0.133 +/- 0.055 l h-1 kg-1 (range 0.073-0.233) and was not significantly different (P greater than 0.05) from that observed in the control group (0.125 +/- 0.025 l h-1 kg-1, range 0.075-0.148).