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Using (-)-indolactam V to induce differentiation of human and mouse embryonic stem cells into pancreatic lineage

• ILV can be used alone, or in combination with growth factors, to direct differentiation of ESCs into pancreatic specification. ILV is effective on human ESC lines and mouse ESC lines.

• ILV is effective on human ESC lines HUES 2, 4 and 8. After 4 days treatment with ILV alone, 27% of HUES 8–derived cells were positively stained by Pdx1 antibody.

• In mouse ESCs that had been induced to form definitive endoderm (DE), after treatment with 300 nM ILV, the percentage of Pdx1-expressing cells increased from 4% to 50%.

• Titration of ILV from 20 nM to 5 mM showed that it functions in a dose-dependent manner. Effector concentration for half-maximum response (EC50) = 142 nM; with the highest efficacy and no detectable toxicity at 300 nM.

• ILV acts synergistically with FGF10, a growth factor involves in pancreatic development. When a population of HUES 8-E cells was treated with a combination of 300 nM ILV and 50 ng/ml FGF10, after 4 days culture, 46% of the cells were positively stained by Pdx1antibody.

Intellectual Property Status: Patent(s) Pending

Applications

A possible application of human embryonic stem cells (hESCs) is to generate new insulin-producing beta cells for cell replacement therapy for diabetics. Stepwise protocol is being explored to differentiate hESCs into functional beta cells. An essential step is the generation of pancreatic progenitors -- cells that express Pdx1 and produce all the cell types of the pancreas. As an alternative to using coculture with other cell types and growth factors as inducers, the laboratory of Douglas Melton uses cell-permeable small molecules as a means to control in vitro differentiation of hESCs. Small-molecule inducers would be less expensive, more easily controlled and possibly more efficient than growth factors in directing differentiation. The Melton's group has identified that (-)-indolactam V (ILV) activates PKC signaling and directs the pancreatic specification of hESCs that have already been committed to the endoderm lineage.

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