Failure of insulin cells to develop in cultured embryonic chick pancreas: A model system for the detection of factors supporting insulin cell differentiation

Abstract

Little being known about factors necessary for insulin cell differentiation, we tested the chance observation that these cells were virtually absent from collagen gel cultures of embryonic avian pancreas in which the other pancreatic endocrine cells were numerous. Five-day dorsal buds stripped of their enveloping mesenchyme were embedded in gel and overlaid by a defined medium containing serum, then cultured for 7 days. Immunocytochemical evaluation showed a very low proportion of insulin cells. Substitution of the gel by a polyamino acid coating slightly increased the proportion. In an attempt to test for ability of insulin cell formation to recover, we transferred explants first cultured in collagen gel to polyamino-acid-coated dishes for a further 7 days. No improvement resulted. In controls grown for 14 days on a polyamino acid coating, insulin cells disappeared completely. We conclude that collagen gel does not support survival and differentiation of chick embryonic insulin cells and that the medium used is lacking in some essential factor(s). Determination of their identity should prove possible by exploitation of this model.

Morphogenesis and differentiation of the avian endocrine pancreas, with particular reference to experimental studies on the chick embryo

“…Hence, it can be inferred that because the number of both acinar and insulin cells was affected, these cell types require something in MF for their differentiation. Similar experiments have not been conducted on avian embryos but it is notable that when chick dorsal pancreatic bud primordia, from which almost all mesenchyme has been removed, are cultured in the absence of a supporting matrix, such as collagen gel or Matrigel, they remain very small (see Andrew et al, 1994).…”

“…In addition to cells that contain the major islet hormones, cells containing a neurotensin-and occasionally a gastrin/cholecystokinin-like peptide have been observed in cultures of chick dorsal pancreatic bud endoderm (Andrew et al, 1994), while in grafts of the chick dorsal bud, endocrine cells ectopic to the pancreas, exhibiting immunoreactivity for neurotensin and motilin, have been observed in rare instances (Andrew et al, 1988). There is, therefore, the potential in avian pancreas for differentiation of endocrine cell types not normally found there.…”

“…With a view to identification of factors specifically required for differentiation of insulin cells, an in vitro system utilising the dorsal pancreatic bud of the chick embryo has been established (see Andrew et al, 1994;Rawdon and Andrew, 1997;Rawdon and Andrew, 1988). This model system enables the nature of the substrate and medium to be manipulated.…”

Regulation of Embryonic Chick Insulin Cells: Effect of Retinoic Acid and Insulin-Like Growth Factor 1

“…RA alone (10 -6 M) was shown to increase the proportion of insulin cells by a factor of 3 [Penny and Kramer, 2000]; the addition of nicotinamide increased the proportion of insulin cells by a factor of 2.5 [Mngomezulu and Kramer, 2000] and IGF-1 by a factor of 1.7 [Rawdon and Andrew, 1998]. None of the results of these studies have approached the in vivo proportion of 34% for insulin cells from 12-day splenic lobe of intact embryos [Andrew et al, 1994]. This may relate to the constant in vivo conditions, in which circulating factors are available and at the correct physiological levels.…”

“…The increased proportions of insulin cells were obtained in a culture system using Matrigel and Ham's F12 medium supplemented with transferrin and selenium. Increased proportions of insulin cells have been achieved by the manipulation of the culture medium and the extracellular matrix [Andrew et al, 1994;Rawdon andAndrew, 1997, 1998;Mngomezulu and Kramer, 2000;Penny and Kramer, 2000]. RA alone (10 -6 M) was shown to increase the proportion of insulin cells by a factor of 3 [Penny and Kramer, 2000]; the addition of nicotinamide increased the proportion of insulin cells by a factor of 2.5 [Mngomezulu and Kramer, 2000] and IGF-1 by a factor of 1.7 [Rawdon and Andrew, 1998].…”