Development of Cytotrophoblast Columns from Explanted First-Trimester Human Placental Villi: Role of Fibronectin and Integrin α5β11

Abstract

Human first-trimester floating mesenchymal villi explanted onto gels of collagen I or Matrigel were observed to undergo de novo development of anchoring sites. These consisted of cytotrophoblast columns that formed by proliferation of stem villous cytotrophoblast cells, as revealed by whole-mount and thin-section microscopy and incorporation of bromodeoxyuridine into DNA. Column formation occurred exclusively at the distal tips of the villi. No column formation was observed in tissue explanted onto agarose. On Matrigel, the developing columns penetrated downwards into the matrix, whereas on collagen I, cytotrophoblast sheets spread across the surface of the gel and merged to form a shell. The developing columnar cytotrophoblast up-regulated integrins alpha1beta1 and alpha5beta1 and produced an extracellular matrix containing oncofetal fibronectin, as in vivo. Function-blocking antibodies were used to investigate the role of the integrin-fibronectin interaction in anchoring villus development on collagen I. Antibodies to fibronectin and the integrin subunits alpha5 and beta1, added at 24 h, all changed the pattern of cytotrophoblast outgrowth. Anti-fibronectin caused cell rounding within the cytotrophoblast sheet and increased the population of single cells at its periphery. Anti-integrin alpha5 caused rounding and redistribution of cells within the outgrowth. In the presence of anti-integrin beta1, cell-collagen interactions within the sheet were destabilized, often leading to the appearance of an annulus of aggregated cells at the periphery. These results show that 1) mesenchymal villi retain the potential to form anchoring sites until at least the end of the first trimester, 2) adhesion to a permissive extracellular matrix stimulates cytotrophoblast proliferation and differentiation along the extravillous lineage, 3) integrin alpha5beta1-fibronectin interactions contribute significantly to anchorage of the placenta to uterine extracellular matrix. We suggest that as the developing placenta ramifies, new sites of anchorage form whenever peripheral villi contact decidua. This process is predicted to contribute to the stability of the placental-decidual interface.

Quantifying trophoblast migration: In vitro approaches to address in vivo situations

“…Such villous explant models involve the culture of small pieces of first trimester placental tissue on type I collagen or Matrigel, resulting in the formation of EVT outgrowths from the tips of anchoring villi in contact with the extracellular matrix substrate. [25][26][27][28][29] In any culture only a proportion of explants produce trophoblast outgrowths, dependent on the culture conditions employed and the tissue gestation. 27,29,30 The formation of these EVT outgrowths represents the first stages of villous cytotrophoblast differentiation into the EVT lineage, and further expansion of the outgrowth can then be used to study EVT migration and/or invasion.…”

“…Traditionally, many villous explant models embedded explants in a thick layer of Matrigel. 26,28,41 In these systems the EVT outgrowths can expand and migrate in 3-dimensions by actively invading through the Matrigel, but the accurate vizualization and quantification of the resulting outgrowth volume can become problematic, 26,28,41 It is also possible to culture villous explants on top of an extremely thin layer of Matrigel (Fig. 1A and B).…”

Adhesion Molecules in Human Trophoblast – A Review. II. Extravillous Trophoblast

“…Cells in the columns upregulate the fibronectin receptor integrin α5β1 [3][4][5][6][7][8][9][10][11], the fibronectin, VCAM-1 and EMILIN-1 receptor α4β1 [12], αVβ3 [12], which binds fibronectin and other RGD-containing ligands in extracellular matrix (ECM), the laminin/collagen receptor α1β1 and the laminin receptor α6β1. They exhibit a reciprocal loss 35 of expression of the laminin receptor α6β4 [4,6,[13][14][15][16].…”

“…Functional studies examining the role of integrins in mediating invasion of primary cytotrophoblasts (CTB) through a basement membrane-like matrix (Matrigel) and interstitial collagen have demonstrated the importance of αvβ3 and α1β1 in promoting migration and of α5β1 in anchorage [5,6,8,12]. Function-blocking antibodies raised against β1 disturb 40 anchorage and reduce column outgrowth when applied to villous explants, and shift the balance of interactions away from cell-matrix and towards cell-cell adhesion [8].…”

Human HLA-G+ extravillous trophoblasts: Immune-activating cells that interact with decidual leukocytes

“…EVT invade decidual tissue and interact with maternal decidual natural killer cells (dNK), decidual macrophages (dMϕ), and decidual T cells (dT) (1,6). EVT are difficult to study due to the low EVT numbers that can be obtained from tissue and the lack of proliferative capacity that precludes their expansion in vitro (7)(8)(9).…”