Noggin, Retinoids, and Fibroblast Growth Factor Regulate Hepatic or Pancreatic Fate of Human Embryonic Stem Cells

Background & Aims

New sources of β cells are needed to develop cell therapies for patients with diabetes. An in vitro, sequential method has been developed to derive pancreatic progenitors, but this technique has not been used for other cell lines. We investigated whether definitive endoderm derived from human embryonic stem (hES) cells might be used to create β cells.

Methods

Five hES cell lines were induced to form pancreatic progenitors and analyzed for pancreas markers. Cells were incubated with a bone morphogenetic protein (BMP) antagonist, retinoids, a Hedgehog antagonist, or fibroblast growth factor (FGF) and phenotypes were analyzed.

Results

Four hES cell lines sequentially generated definitive endoderm, primitive gut, and posterior foregut equivalents, as described previously. However, functional hepatocytes, rather than pancreas progenitors, developed. Consistent with liver development, FGF and BMP signaling pathways were involved in this process; their inhibition disrupted hepatocyte differentiation. During early stages of development, exposure of cells to noggin and retinoid acid, followed by FGF10, generated pancreatic cells (PDX1+; 50%−80%) that coexpressed FOXA2, HNF6, and SOX9.

Conclusions

These findings demonstrate the combined functions of endogenous BMP and supplemented FGF in inducing differentiation of hepatocytes from hES cells and the ability to shift developmental pathways from hepatic to pancreatic cell differentiation. Although additional signals appear to be required for full specification of PDX1⁺ early pancreatic progenitors (via PTF1a and NKX6.1 coexpression), these findings indicate the signaling pathways required for differentiation of bipotential progenitors.

Keywords: Albumin, α-Fetoprotein, Insulin, β Cell

Abbreviations used in this paper: AFP, α-fetoprotein, ALB, albumin, BMP, bone morphogenetic protein, DE, definitive endoderm, FGF, fibroblast growth factor, hES cell, human embryonic stem cell, PBS, phosphate-buffered saline, PFG, posterior foregut, PGT, primitive gut tube, PPP, PDX1-positive progenitor, QRT-PCR, quantitative reverse transcription polymerase chain reaction, RA, retinoic acid