Generation of hepatocytes and pancreatic endocrine cells from human induced endodermal progenitor like cells

Acknowledgments: This work is funded by Dutch Diabetes foundation, Netherlands and funds from KU Leuven. Disclosures: Catherine Verfaillie is an advisor to ReGenesys.Rangarajan Sambathkumar, Philip Roelandt1, Manoj Kumar1, Ana Rita Mestre Rosa1, Fanos Tadessa Woldemariyam1, Renate Akkerman1, Sumitava Dastidar2, Qing Cai1, Eric Kalo1, Valerie Roobrouck3, Satish Khurana1, Marijke Faas5, Paul de Vos5, Catherine M Verfaillie1. New sources of cells are needed for both therapy of diabetes (beta cells) and creation of hepatocytes (for toxicology and metabolization studies). One possibility are induced pluripotent stem cells generated from fibroblasts and then differentiated towards the lineage of interest. One drawback, definitely for clinical therapies is the possible persistence of pluripotent cells that might form teratomas. An alternative approach is to de-differentiate somatic cells to an intermediate progenitor, such as endodermal progenitors, which can then be committed to beta-cells and / or hepatocytes. We previously reported that using 16 TFs, human multipotent adult progenitor cells (MAPCs) could be transdifferentiated in cells with epithelial morphology, that could be expanded long term and expressed (mes)endodermal genes. However, more mature endodermal genes were also expressed including Albumin (ALB). We therefore tested a combination of 14 TF, which now resulted in iENDO cells; expandable cells with (mes)endodermal endogenous gene expression but without mature endodermal gene expression. Using adapted protocols for pancreatic endocrine cell differentiation and hepatocyte differentiation, we demonstrated that 14 TF iEndo cells could be committed to endocrine pancreatic endoderm, expressing PDX1, NGN3, PAX4, NKX2.2, NEUROD1, MAFA and MAFB, but not mature beta cell characteristics (no Insulin expressed). During hepatocyte differentiation, hepatoblast genes such as AFP, ALB, AAT were induced. Inability to create fully committed progenitors at this point may be because the differentiation conditions are not yet fully optimized. A second possibility is that the OCT4 transgene used in the preprogramming remains expressed. Currently we are testing if doxycycline inducible overexpression of OKSM combined with the other 10 TFs may support full differentiation towards beta cells and hepatocytes. Preliminary results suggest that upon transplantation of undifferentiated 14TFs iENDO cells under the kidney capsule of SCID mice, cells are differentiating to the hepatocyte lineage In vivo. Studies wherein iENDO cells differentiated to endocrine pancreatic cells are evaluated following grafting in vivo are pending.status: publishe