Endoderm Differentiation from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) provide a virtually unlimited raw material to derive and engineer mature cell types with therapeutic value, including cell transplantation, disease modeling and drug screening. The first step to differentiate hPSCs into such cell types involves specifi cation towards one of the three main embryonic cell populations, ecto-, endo- and mesoderm. Efficient induction into the correct lineage is critical to the success of subsequent differentiation steps and to the final yield of desired cells. Here we describe methods to generate definitive endoderm (DE), the progenitor cell population for such tissues as the thymus, liver, pancreas, stomach and intestine. In addition, we will provide methods to characterize and monitor the effi ciency of DE differentiation, including expression of DE markers at the gene and protein level. Flow cytometry based methods described in this chapter can also be extended to isolate and purify cells with DE properties. Such enrichment strategies are useful to eliminate undesired cell populations, especially undifferentiated hPSCs, which harbor the potential risk for seeding tumors upon transplantation. Several of the methods for the manipulation of hPSCs and for their analysis outlined here are of general utility and are applicable to other hPSCs derivative cell populations.