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.