De differentiatie van geïnduceerde endodermale progenitorcellen, afgeleid van MAPCs, naar humane hepatocyten en pancreascellen.

New and renewable sources of cells are needed for cell based therapies of liver diseases and type 1 diabetes (T1D). Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or β-cells. However, human MAPCs have less plasticity than pluripotent stem cells (PSCs), as their ability to generate endodermal cells is not robust. The creation of induced pluripotent stem cells (iPSC) has demonstrated that reprogramming of somatic cells to a pluripotent fate is possible. Reprogramming can occur by the introduction of key pluripotency transcription factors (TFs) as well as epigenetic changes. This finding has sparked interest in direct reprogramming of one somatic cell into another, using lineage specific transcription factors (TFs) with or without epigenetic modifiers. In a first set of studies, I studied whether 14 transcription factors (TFs) can reprogram MAPCs to induced endodermal progenitor cells (iENDO cells), defined as cells that can be long-term expanded and differentiated to both hepatocyte- and endocrine pancreatic like cells. I demonstrate that iENDO cells can be generated by transduction with 14TFs. iENDO cells expressed endodermal markers but nor mature endodermal and pluripotency gene expression. 14TF-iENDO cells could be expanded for at least 20 passages, differentiated in vivo spontaneously to hepatocytes, endocrine pancreatic cells, as well as to gut tube-like cells when grafted in immunodeficient mice. Furthermore iENDO cells can be specifically differentiated in vitro into hepatocyte or endocrine pancreatic-like cells using 3D organoid cultures. However, the pluripotency TF OCT4, which is one of the TFs used for inducing iENDO cells was not silenced, which might contribute to the incomplete differentiation of iENDO cells to mature hepatocytes and endocrine pancreatic β-cells in vitro, and to endodermal tumor formation in vivo. To assess the in vivo maturation of iENDO cells differentiated to hepatocyte-like cells in vitro, we transplanted the iENDO-derived hepatocyte like cells organoids under the kidney capsule of immunocompromised mice. After 2 months, grafts of hepatocyte like cell progeny from iENDO cells appeared to have matured further, with more cells expressing albumin (ALB) and fewer AFP. Although full quantification still needs to be done, we did not observed any tumor formation. All grafts secreted albumin. iENDO differentiated pancreatic endocrine cells have been transplanted in diabetic or non-diabetic nude mice studies; the function and phenotype of the grafts is currently being evaluated. These studies provide evidence that reprogramming of adult stem cells to an endodermal intermediate progenitor, which can be expanded and differentiated to multiple endodermal cell types might be a valid alternative for the use of PSCs for creation of endodermal cell types. In parallel I tested if epigenetic modifiers, namely the histone deacetylase inhibitor (HDACi) Trichostatin (TSA) and the DNA methyltransferase inhibitor (DNMTi) 5-azacytdine (5AZA) can reprogram primary human fibroblasts to an endodermal and pancreatic cell fate. I demonstrate that TSA combined with chromatin-remodeling medium (CRM) induced expression of a number of definitive endoderm and early and late pancreatic marker genes. When CRM was omitted, endoderm/pancreatic marker genes were not induced. However, treatment with 5AZA did not affect gene expression changes, and when 5AZA was combined with TSA, no further increase in endoderm, pancreatic endoderm, and endocrine gene expression was seen beyond levels induced with TSA alone. Interestingly, TSA-CRM did not affect expression of pluripotency and hepatocyte genes but induced some mesoderm transcripts. Upon removal of TSA-CRM, the endoderm/pancreatic gene expression profile returned to baseline. Our findings underscore a role of epigenetic modification in transdifferentiation of one somatic cell into another. However, full reprogramming of fibroblasts to β-cells will require combination of this approach with TF overexpression and/or culture of the partially reprogrammed cells under β-cell specific conditions.nrpages: 192status: publishe