Proliferation and differentiation of porcine inner cell mass and epiblast in vitro

The proliferation rate and differentiation state were investigated in porcine inner cell masses (ICMs) and epiblasts in vitro. ICMs isolated from early blastocysts (Day 7 of pregnancy) and epiblasts isolated from preelongated blastocysts (Day 11 of pregnancy) were cultured for up to 5 days in the presence of human leukemia inhibitory factor (hLIF) (1000 U/ml). The proliferation rate was evaluated by determination of the percentage of cells in S-phase. The differentiation state was determined by studying the expression of the stage-specific embryonic antigen-1 (SSEA-1), a marker for undifferentiated murine embryonic stem (ES) cells, and the expression of laminin and cytokeratins 8/18, markers of ES cell differentiation. The staining pattern showed that freshly collected Day 11 epiblasts appeared undifferentiated but rapidly lost this characteristic in vitro. A decrease in the proliferation rate was also observed during culture. This decrease was reduced in the presence of high concentrations of hLIF (optimal concentrations: 5000 U/ml). Conversely, treatment of Day 11 epiblast cells with retinoic acid, an agent known to induce differentiation in murine ES cells, caused a dramatic decrease in the proliferation rate in vitro. In contrast to Day 11 epiblasts, Day 7 ICMs expressed SSEA-1 in vitro and showed a higher proliferation rate (p < 0.01). However, their proliferation rate also decreased during culture and following trypsinization. These results indicate that the undifferentiated characteristics of Day 7 ICMs are more likely to be maintained in vitro than are those of Day 11 epiblasts, which are rapidly committed into early differentiation.