232 SERUM-FREE DERIVATION OF CAT EMBRYONIC STEM-LIKE CELLS DERIVED FROM IN VIVO-PRODUCED BLASTOCYSTS ON CAT EMBRYONIC FIBROBLAST FEEDERS

Abstract

Fetal bovine serum (FBS) has usually been used as a component of the medium for the culture of embryonic stem (ES) cells. However, FBS contains undefined factors, which promote cell proliferation and occasionally stimulate differentiation of ES cells. To establish an effective culture system for the derivation and maintenance of cat ES cells, we compared the effects of Knockout^TM Serum Replacement (Invitrogen, Seoul, South Korea)-supplemented medium (KSR-medium) and FBS-supplemented medium (FBS-medium) on the attachment and formation of primary ES-like cell colonies of isolated inner cell mass (ICM) derived from in vivo-produced blastocysts on mitotically inactivated cat embryonic fibroblast (CEF) feeders. An inseminated domestic female cat underwent ovariohysterectomy at Day 7 post-insemination, after which blastocysts were flushed from the uterine horns. Inner cell mass (ICM) was mechanically isolated from the in vivo-matured blastocysts (n = 22) and cultured in KSR-medium or FBS-medium on a CEF feeder layer at 39°C, 5% CO₂ in air. KSR-medium or FBS-medium consisted of knockout^TM DMEM supplemented with 20% KSR or FBS, 100 IU mL^-1 penicillin/streptomycin, 2 mM l-glutamine, 1× MEM non-essential amino acids, 100 µM ²-mercaptoethanol, and 1000 IU mL^-1 recombinant mouse LIF. The results indicate that incidence of primary ICM attachment (91.6 ± 14.4 vs. 55.5 ± 9.6% in KSR- and FBS-medium, respectively; P < 0.05) and ES-like cell colony formation (63.8 ± 12.7 vs. 27.7 ± 4.8% in KSR- and FBS-medium, respectively; P < 0.05) is significantly affected by the ES cell culture medium. However, cat ES-like cells in KSR-medium did not actively proliferate in the primary culture. The number of cat ES-like cells in a colony after 5 days of culture was significantly higher in FBS-medium than in KSR-medium (749 ± 82 vs. 383 ± 55% in FBS- and KSR-medium, respectively; P < 0.05). A total of 10 ES-like cell colonies were formed in KSR-medium (n = 7) and FBS-medium (n = 3), and these expressed high levels of alkaline phosphatase activity (AP). Immunostaining analysis was positive for the ES cell-markers, Oct-4 and SSEA-3. To our knowledge, this is the first reported isolation of cat ES-like cells on CEF feeders in the absence of serum. A culture system of embryo-derived stem cells using serum-free medium may make it possible to directly examine the effects of factors added to culture medium on isolation and maintenance of cat ES cell lines in vitro.

This work was supported by KOSEF (grant # M105250100 01-05N2501-00110).