Isolation, culture, and differentiation of echinoid primary mesenchyme cells

Methods are described for isolation and culture of primary mesenchyme cells from echinoid embryos. Ninety-five percentpure primary mesenchyme cells were isolated from early gastrulae ofStrongylocentrotus purpuratus, exploiting the biological segregation of these cells within the blastocoel. When cultured, more than 90% of the isolated cells reached the differentiated state, spicule formation, in synchrony with in vivo controls. Isolated primary mesenchyme cells were cultured with and without various cellular and acellular components of normal embryos in order to study the potential involvement of these components in the morphogenesis of the primary mesenchyme. Our data indicate that: 1. primary mesenchyme cells lack the ability to form the annular pattern of the primary mesenchymal ring autonomously; 2. they autonomously produce spicules of a characteristic morphology that differs from that of embryonic spicules; 3. morphogenesis of the primary mesenchyme is not affected by association with embryonic basal lamina, blastocoel matrix, or loosely aggregated epithelial cells, or by close confinement of each set of primary mesenchyme cells within the blastocoelar space; and 4. reaggregated, tightly associated epithelial cells can promote normal primary mesenchyme ring formation, and modify the primary mesenchyme-intrinsic spicule pattern to produce more normal spicule forms.