286 ESTABLISHMENT OF PORCINE EMBRYONIC STEM CELL-LIKE CULTURES USING DIFFERENT MEDIA

Abstract

Porcine embryonic stem cells (pESC) have the potential of becoming an invaluable model for cell-based therapy. However, stable pESC lines are still lacking. We aimed at evaluating various culture media containing growth factors known to support the growth of murine and human ESC for generation of pESC. A total of 186 zona pellucida enclosed (ZPE) and 8 zona pellucida hatched (ZPH) blastocysts were isolated by flushing of sow uteri 5 days post insemination. A total of 122 of the ZPE blastocysts were selected for culture on mouse embryonic fibroblasts (MEF) using either culture of whole blastocyst or immunosurgery. Six types of media were tested: (1) Pig ESC medium [pESCm; DMEM/F10 (1:1), 10% Knockout Serum (KSR), 5% Fetal Bovine Serum and 0.1% leukaemia inhibitory factor (LIF)], (2) pESCm supplemented with 20 ng mL^–1 stem cell factor (SCF) (pESCm+SCF), (3) pESCm supplemented with 4 ng mL^–1 basic fibroblast growth factor (bFGF; pESCm+bFGF), (4) Human ESC medium (hESCm; Knockout DMEM, 15% KSR and 4 ng mL^–1 bFGF), (5) hESCm supplemented with 0.1% LIF (hESCm+LIF) and (6) a commercial LIF containing medium (Resgro; Millipore A/S, Copenhagen, Denmark) used for derivation of murine ESC. At Day 7 of culture, outgrowth colonies (OC) were morphologically examined and attachment rates as well as number of ES-like colonies were noted. ES-like colonies were defined as delineated colonies containing cells with a high nucleo-cytoplasmic ratio and one or two distinct nucleoli. This morphology was verified by nuclear immunocytochemical OCT4-staining (Santa Cruz Biotechnology, Santa Cruz, CA, USA) upon 4% paraformaldehyde fixation. Manual passaging was performed on Day 8 to 10 and RT-PCR was performed on pieces of colonies to evaluate OCT4, Nanog and SOX2 expression. Attachment rates were similar across media groups using whole blastocyst culture and immunosurgery (46 v. 39%, respectively) as were the rates of ES-like OC (80 v. 74%). The highest attachment rates were obtained in pESCm+SCF (60%), hESCm+LIF (55%) and pESCm+bFGF (54%), whereas rates were significantly lower in pESCm (36%), Resgro (33%) and hESCm (22%). High numbers of ES-like colonies were obtained in hESCm (100%) and pESCm+bFGF (100%) though not significantly different from pESCm (89%), hESC+LIF (83%) and pESCm+SCF (66%). Culture in Resgro medium, on the contrary, resulted exclusively in differentiated OC. The maximum number of passages was obtained in pESCm (7 passages) and hESCm (4 passages) after which the colonies started to differentiate into an epithelial-like morphology or became quiescent. RT-PCR analyses showed that most of the ES-like OC were positive for all the pluripotency markers tested, but the passages were, in general, either negative or inconsistently positive for Oct4, Nanog and/or Sox2. In conclusion, supplementation of media with growth factors used for culture of human and murine ESC had a positive effect on the attachment rate of pESC, whereas an effect on the rate of ES-like OC was lacking. The commercial murine ESC medium, Resgro, did not yield ES-like OC at all. Further optimization is needed in order to maintain pluripotency after passage.