HNF-6-independent differentiation of mouse embryonic stem cells into insulin-producing cells

Abstract

Aims/hypothesis. Embryonic stem cells, when grown as embryoid bodies, spontaneously generate insulinproducing cells which could be used in therapy of diabetes mellitus, provided that their selection and differentiation are optimized. To achieve such optimization, one needs to know whether the differentiation of cells in embryoid bodies mimicks that of pancreatic beta cells in embryos. To address this question we verified if the differentiation of the insulin-producing cells in embryoid bodies requires Hepatocyte Nuclear Factor-6 (HNF-6), a transcription factor known to control pancreatic endocrine differentiation in embryos. Methods. We generated mouse Hnf6−/− embryonic stem cells and grew them as embryoid bodies. The expression of HNF-6, insulin, and transcription factors that are regulated by HNF-6 in developing pancreas was compared in wild-type and Hnf6−/− embryoid bodies. Results.No difference was observed in the expression of insulin between wild-type and Hnf6−/− embryoid bodies. In both cases insulin was expressed in the outer layer of cells, which is similar to the visceral endoderm. In wild-type embryoid bodies HNF-6 was transiently expressed in the outer layer of cells, but was not co-expressed with insulin. The expression of genes that are targets of HNF-6 in developing pancreas was unaffected in Hnf6−/− embryoid bodies. Conclusion/interpretation. In contrast to the development of pancreatic beta cells, the differentiation of insulin-producing cells in embryoid bodies did not require HNF-6. Thus, the differentiation mechanism of insulin-producing cells in embryoid bodies differs from that of the beta cells and it is likely to resemble that of insulin-producing cells in the visceral endoderm. [Diabetologia (2003) 46:378-385]

Committing Embryonic Stem Cells to Early Endocrine Pancreas In Vitro

“…However, previous ES cell culture studies [25,[39][40][41][42][43][44] did not address nor specify the extent of endoderm and pancreatic differentiation in their cultures. Our results demonstrated that the S EB-derived population expressed both insulin I and II genes, as well as glucagon, pdx-1, and Sox17.…”

“…Recent efforts have been directed at the generation of insulin-producing cells using the ES-EB culture system [25,[39][40][41][42][43][44] to provide islet cells for transplantation and cure of type I diabetes. However, previous ES cell culture studies [25,[39][40][41][42][43][44] did not address nor specify the extent of endoderm and pancreatic differentiation in their cultures.…”

Stem cell therapy for diabetes: do we need to make beta cells?

“…A large number of reports have now demonstrated that ES cells can differentiate into cells with an insulin-expressing phenotype, either by genetic manipulation or by permitting spontaneous differentiation followed by culture under selective conditions (Soria et al 2000, Assady et al 2001, Lumelsky et al 2001, Hori et al 2002, Shiroi et al 2002, Blyszczuk et al 2003, Moritoh et al 2003, Miyazaki et al 2004, Segev et al 2004, Sipione et al 2004. In common with studies using tissue stem cells, the cellular identity of these insulin-expressing cells is uncertain and with the lack of specific, easily identifiable markers of a mature -cell, the possibility remains that these cells are not fully mature -cells but instead are a phenotypically similar population of cells, perhaps of neuroectodermal (Lumelsky et al 2001, Burns et al 2003 or extra-embryonic (Houard et al 2003) origin.…”

“…There is some evidence that it may be possible to employ conscious design to arrive at the same end-point by a less circuitous route. Thus, it has been suggested that the pathways of -cell differentiation in vitro may differ significantly from those in vivo (Houard et al 2003) and mouse ES cells are reported to differentiate into endocrine cells without PDX-1 (pancreatic duodenal homeobox-1) expression (Moritoh et al 2003), although this is essential in vivo (Scharfmann 2000). It is also possible that current in vitro differentiation protocols do not generate -cells, but, instead, cells that have some phenotypic and functional similarity to authentic -cells.…”

“…Visceral endoderm develops initially in a similar manner to embryonic endoderm, and primitive endodermal cells are localised to the periphery of EBs (embryoid bodies) during differentiation of mouse ES cells (Murray & Edgar 2001). It has, therefore, been suggested that the insulin-expressing cells generated during EB formation are from visceral endoderm, since they are located almost exclusively in the outer layer of the EB (Houard et al 2003). In the absence of specific and highly expressed markers for authentic -cells it is difficult to determine unambiguously the origin of insulin-expressing cells generated in vitro from pluripotent progenitor populations.…”