Background and Aims Induced pluripotent stem cells (iPSCs) provide an important tool for the generation of patient‐derived cells, including hepatocyte‐like cells, by developmental cues through an endoderm intermediate. However, most iPSC lines fail to differentiate into endoderm, with induction resulting in apoptosis. Approach and Results To address this issue, we built upon published methods to develop an improved protocol. We discovered that doxycycline dramatically enhances the efficiency of iPSCs to endoderm differentiation by inhibiting apoptosis and promoting proliferation through the protein kinase B pathway. We tested this protocol in >70 iPSC lines, 90% of which consistently formed complete sheets of endoderm. Endoderm generated by our method achieves similar transcriptomic profiles, expression of endoderm protein markers, and the ability to be further differentiated to downstream lineages. Conclusions Furthermore, this method achieves a 4‐fold increase in endoderm cell number and will accelerate studies of human diseases in vitro and facilitate the expansion of iPSC‐derived cells for transplantation studies.

Induced pluripotent stem cells (iPSCs) provide an important tool for the generation patientderived cells including hepatocyte-like cells via developmental cues through an endoderm intermediate. However, most iPSCs fail to differentiate into endoderm, with induction resulting in apoptosis. To address this issue, we built upon published methods to develop an improved protocol with our discovery that doxycycline dramatically enhances the iPSC to endoderm differentiation efficiency by inhibiting apoptosis and promoting proliferation via the AKT pathway.We tested this new protocol in more than 70 iPSC lines with consistent formation of complete sheets of endoderm in 90%. Endoderm generated by our method achieves similar transcriptomic profiles, including FOXA2, HNF1, CXCR4, and SOX17 positive cells, and the ability to be further differentiated. Furthermore this method achieves a four-fold increase in endoderm cell number and will accelerate studies of human diseases in vitro and facilitate the expansion of iPSC-derived cells for transplantation studies.

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Induced pluripotent stem cells (iPSCs) provide an important tool for the generation of patient‐derived cells including hepatocyte‐like cells via developmental cues through an endoderm intermediate. However, most iPSC lines fail to differentiate into endoderm, with induction resulting in apoptosis. To address this issue, we built upon published methods to develop an improved protocol. We discovered that doxycycline dramatically enhances the iPSC to endoderm differentiation efficiency by inhibiting apoptosis and promoting proliferation via the AKT (protein kinase B) pathway. We tested this new protocol in more than 70 iPSC lines with consistent formation of complete sheets of endoderm in 90%. Endoderm generated by our method achieves similar transcriptomic profiles, positive immunohistochemistry for endoderm protein markers, and the ability to be further differentiated. Furthermore this method achieves a four‐fold increase in endoderm cell number and will accelerate studies of human diseases in vitro and facilitate the expansion of iPSC‐derived cells for transplantation studies.