Retinoid acid attenuates amylase expression in human embryonic stem cells-derived endoderm and improves hepatic fate.

Background and Aims: Human embryonic stem cell (hESC) derivatives are now regarded as potential surrogates for cell therapy. Many patients suffering from various diseases would benefit from it provided the in vitro differentiation of functional cells is well mastered. As far as diabetes is concerned, hESC have been coaxed towards insulin-producing cells, but the differentiation efficiency and functional status of these cells remain to be improved. In an attempt to design a better protocol, we sequentially cultured hESC in the presence of several growth factors and chemicals previously described as improving pancreas differentiation.

Materials and Methods: The hESC line VUB07 generated and characterized in our institute was maintained undifferentiated on inactivated mouse embryonic fibroblasts and differentiated into definitive endoderm by treatment with ActA (50 ng/ml) and Wnt3a (25 ng/ml), with or without BMP4 (8 ng/ml) and ITS supplement (1x) for 4 days. Retinoic acid (2.5-10 mmol/l) and ITS (1x) were added for 2 days and the cells further maintained in low FCS (2%), FGF2 (5 ng/ml), ActB (10 ng/ml) and KAAD-Cyclopamine (250 nmol/l) for 4 days. During the last 5 days, Cyclopamine and ActB were replaced by Nicotinamide (10 mmol/l). Pancreatic and hepatic markers were analyzed at each stage by immunocytochemistry and PCR.

Results: Definitive endoderm was induced in this system as evaluated by Sox17 and Foxa2 co-expression (up to 60%) on day 4. Analysis of pancreatic markers indicated that in the absence of retinoic acid treatment, a large majority of differentiated cells (50-60%) strongly express amylase and low levels of Pdx1. On the contrary, addition of retinoic acid (days 4 to 6) attenuated amylase expression in the progenies, but did not result in a significant increase in Pdx1 expression. Furthermore, retinoic acid considerably improved the generation of albumin-expressing hepatocyte-like cells organized in colonies that co-expressed Foxa2, in contrast to the rare albumin-positive cells observed in its absence. The role of retinoic acid in this process and in hepato-pancreas development is further discussed.

Conclusions: Sequential exposure of hESC to growth factors and chemicals would allow controlling their differentiation towards desired progenies such as pancreatic cells needed in diabetes cell therapy. hESC-derived definitive endoderm was easily converted to amylase-expressing cells within 2 weeks, whereas its early treatment with retinoic acid shifted the progenies to a hepatic fate. This negative effect of retinoic acid on exocrine pancreas differentiation is concordant with data from mouse studies, but does not support the established requirement of retinoic acid for pancreas induction from the endoderm.