Insect midgut epithelium in vitro: an insect stem cell system

Mixed cell cultures and stem cell cultures were prepared from midguts of Manduca sexta pharate fourth instar and mid-wandering fifth instar larvae. An extract prepared from the fat body was able to promote stem cell proliferation and affect differentiation in a dose-dependent manner. DNA synthesis activity was confirmed by use of [ 3H]thymidine. Immunohistological localization of bromodeoxyuridine (BrdU), a thymidine analog, indicated that dividing stem cells incorporated the label. In many cases, one of the daughter cells incorporated the label while the other did not; often this daughter appeared morphologically different from its sister cell. These results implied that one of the sister stem cells remained as a proliferating stem cell while the other sister was committed to differentiate. Studies strongly suggest that these midgut cell cultures comprise a true stem cell system. Cell-free conditioned medium from cultures of differentiating pharate fourth instar midgut cells induced development of larval columnar cells from mid-wandering fifth instar midgut stem cells. Conversely, conditioned medium from differentiating cultures of mid-wandering fifth instar midgut induced development of mid-wandering fifth instar low columnar cells from midgut stem cells isolated from pharate fourth instar larvae. Therefore, it appears that differentiating cells produce soluble cytokines which direct specific modes of differentiation by M. sexta stem cells.