Factors affecting cellular outgrowth from porcine inner cell masses in vitro

Abstract

During early embryonic development, endodermal cells leave the inner cell mass (ICM) and migrate over an extracellular matrix located on the blastocoelic side of the trophectoderm to form extraembryonic endoderm. Two experiments were conducted to evaluate factors supporting porcine endodermal cell migration in vitro. In Exp. 1, porcine ICM were cultured on matrices of collagen IV, fibronectin, or laminin. Percentages of ICM generating cellular outgrowth on fibronectin (5/11; 45%) and laminin (4/10; 40%) were similar (P > 0.10); however, collagen IV (0/10; 0%) failed (P < 0.05) to support cellular outgrowth. Inner cell mass and outgrowth areas and numbers of cells in outgrowths were similar (P > 0.10) for fibronectin and laminin, and increased (P < 0.05) with time in culture. In Exp. 2, ICM were cultured on fibronectin or laminin in medium containing 0 or 500 microg/mL of the inhibitory tripeptide, arg-gly-asp (RGD), or on laminin in medium containing 0 or 10 microg/mL recombinant human tissue inhibitor of matrix metalloproteinases-2 (rhTIMP-2). Inner cell mass and outgrowth areas and numbers of cells in the outgrowths for ICM cultured on fibronectin did not differ (P > 0.10) due to the presence of RGD. Inner cell masses cultured on laminin in medium containing 500 microg/mL RGD had fewer cells in the outgrowths and slower rates of cell migration compared with 0 microg/mL (P < 0.05). No differences (P > 0.10) in ICM and outgrowth areas and numbers of cells in the outgrowths were observed for ICM cultured on laminin in medium containing 0 or 10 microg/mL rhTIMP-2. Both fibronectin and laminin supported porcine ICM outgrowth in vitro; however, because outgrowth on fibronectin was not inhibited by RGD, endodermal cells must express an integrin that recognizes an alternative sequence in fibronectin. Cell migration on laminin was inhibited by RGD, suggesting either RGD competes with laminin for binding sites on endodermal cells or binding RGD alters endodermal cell migration on laminin. Because rhTIMP-2 had no effect on cell outgrowth, porcine ICM do not appear to be responsive to the proliferative effects of rhTIMP-2.