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IDE1 and IDE2, small molecule inducers of definitive endoderm from mouse and human embryonic stem cells

The laboratory of Douglas Melton has identified two novel cell-permeable small molecules, IDE1 and IDE2, that direct differentiation of embryonic stem cells (ESCs) into the endodermal lineage. These compounds induce nearly 80% of ESCs to form definitive endoderm, a higher efficiency than that achieved by Activin A or Nodal, commonly used protein inducers of endoderm. The chemically induced endoderm expresses multiple endodermal markers, can participate in normal development when injected into developing embryos, and can continue to differentiate into pancreatic progenitors. IDE1 and IDE2 function in part via activation of TGF-beta signaling, as evident by Smad2 phosphorylation. The application of small molecules to differentiate mouse and human ESCs into endoderm represents a step toward achieving a reproducible and efficient production of desired ESC derivatives.



• IDE1 or IDE2 can be used to direct differentiation of ESCs into definitive endoderm. IDE1 and IDE2 are effective on human and mouse ESC lines.

• IDE1 and IDE2 are products of de novo chemical synthesis and come from a library of putative HDAC inhibitors.

• Titration of IDE1 and IDE2 from 50 nM to 5 mM showed that they function in a dose-dependent manner (EC50 = 125 nM for IDE1 and EC50 = 223 nM for IDE2), with the highest efficiency and no toxicity in the 250–800 nM range.

• At optimal concentration, IDE1 and IDE2 induces the expression of Sox17 (an endodermal marker) in 80% and 72% of mouse ESCs, respectively, after 6 days of treatment. More than 95% of compound-induced Sox17+ cells coexpress FoxA2, an essential gene for the development of definitive endoderm.

• IDE1 and IDE2 also induce endoderm from human ESCs (HUES). Treatment with 100 nM IDE1 or 200 nM IDE2 for 4 days leads to Sox17 expression in 62% or 57% of HUES cells, respectively. More than 90% of Sox17+ cells also coexpressed FoxA2.

Intellectual Property Status: Patent(s) Pending

Applications

An essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Endodermal cell derivatives, including lung, liver, and pancreas, are of interest for regenerative medicine, but efforts to produce these cells have been met with only modest success. As an alternative to using coculture with other cell types and growth factors as inducers, we use cell-permeable small molecules as a means to control in vitro differentiation of embryonic stem cells (ESCs). Small-molecule inducers would be less expensive, more easily controlled and possibly more efficient than growth factors in directing differentiation.

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