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 man ner. DNA synthesis activity was confirmed by use of [H-3]thymidine. Im munohistological 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 th e other did not; often this daughter appeared morphologically differen t from its sister cell. These results implied that one of the sister s tem cells remained as a proliferating stem cell while the other sister was committed to differentiate. Studies strongly suggest that these m idgut cell cultures comprise a true stem cell system, Cell-free condit ioned medium from cultures of differentiating pharate fourth instar mi dgut cells induced development of larval columnar cells from mid-wande ring fifth instar midgut stem cells. Conversely, conditioned medium fr om differentiating cultures of mid-wandering fifth instar midgut induc ed development of mid-wandering fifth instar low columnar cells from m idgut stem cells isolated from pharate fourth instar larvae. Therefore , it appears that differentiating cells produce soluble cytokines whic h direct specific modes of differentiation by M. sexta stem cells.