In vitro differentiation of isolated stem cells from the midgut of Manduca sexta larvae

Isolated spherical stem cells from midguts of pharate fourth-instar larvae of Manduca sexta proliferated in vitro in the presence of 1 ng ml-1 20-hydroxyecdysone and co-cultured fat body tissue or cell-free fat body extract from M. sexta, Lymantria dispar or Heliothis virescens. In this environment, the stem cells were able to undergo mitosis and increase in number. However, stem cells were only able to differentiate to mature goblet and columnar cells when cell-free conditioned medium, taken from midgut cell cultures containing mature cells as well as stem cells and differentiating forms, was introduced into the culture medium. The presence of early and mature goblet cells, lying randomly on their sides, suggested that cell polarity developed in vitro as an intrinsic property of individual cells rather than with reference to an external inductive material. The differentiation factor (or factors) from the conditioned medium appears to include a heat-stable, peptide-like molecule of 10 kDa or less.