Proliferation and Differentiation of Porcine Inner Cell Mass and Epiblast in Vitro1

Abstract

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.

Pig Epiblast Stem Cells Depend on Activin/Nodal Signaling for Pluripotency and Self-Renewal

“…This observation correlates with observations in mEpiSC that show no AP activity [ 6 ]. Surface marker expression shows that mEpiSC are SSEA-1-positive [ 6 , 19 ] and similar observations were reported for pig epiblasts [ 20 ]. We detected strong SSEA-1 expression in cultured epiblasts and weak or no expression of SSEA-4 ( Fig.…”

“…We show that pEpiSC can reactivate the specifi c germ cell markers DAZL and VASA after induction with BMP-4. The capacity of BMP-4 to induce germ cells has been shown in vivo in the mouse [ 40 ] and in vitro with hESC [ 20 ]. Thus, our results with pEpiSC show that the role of BMP-4 as an inducer of the germ cell lineage is conserved.…”

“…5B ). BMP-4 is also effective in inducing differentiation of hESC to germ cell precursors [ 20 ]. Using a similar protocol to that used for hESC, we tested whether germ cell markers were induced from BMP-4-treated pEpiSC.…”

Ultrastructure of the embryonic stem cells of the 8‐day pig blastocyst before and after in vitro manipulation: Development of junctional apparatus and the lethal effects of PBS mediated cell–cell dissociation

“…Similarly, cortical microfilament bundles running between apical junctional complexes are present in the epiblast cells of the chicken primitivestreak-stage blastoderm, a stage where gastrulation is rapidly ensuing (Vakaet and Vanroelen, 1982). Also, a study of differentiation markers showed an undifferentiated state of freshly isolated pig epiblasts taken from 7-and 11-day embryos, and then the differentiation of the epiblast cells within only a few days of culture (Wianny et al, 1997). Thus, it is possible that the appearance of microfilament bundles, cortical and perinuclear, within the cultured pig epiblast cells are differentiation events.…”

Putative Embryonic Stem Cell Lines from Pig Embryos

“…This approach has also been successfully used in the pig [6,20,21,23,28,29,37,38]. Wianny et al [25] developed another immunosurgical method when using a monoclonal antibody SN1/38 specifically raised against porcine trophectodermal cells [50]. Other methods used include treatment with calcium ionophore [51], microsurgery [22,24,32,48] and a digestive method using 0.25% Trypsin/EDTA [30,31].…”