Human Embryonic Stem Cells: Directed Differentiation into Posterior Foregut Endoderm and a Functional Assay for Definitive Endoderm
Abstract: A decade has passed since the first human embryonic stem cell (hESC) line was established, providing immense hope in curing diseases by cell replacement therapy. For instance, in the pancreas, the insulin-producing β-cells are damaged in diabetes patients, and a successful replacement of those cells could provide a cure. These cells can be replaced by organ donation, but owing to a profound scarcity of organ donors, there is a high demand in alternative sources of β-cells. By recapitulating the in vivo developmental pathway, extensively studied in various animal models, it has been demonstrated that hESC can be directed towards a β-cell fate. A complex network of signaling molecules derived from various tissues play multiple roles in the induction events that take place when directing fully pluripotent cells into their different cell fates in vivo, fates that can be followed by analyzing gene and protein expression profiles. The derivation into functional definitive endoderm (DE), the germ layer giving rise to lungs, intestines, liver and pancreas, is a prerequisite for further development towards a pancreatic fate. This thesis covers directed differentiation of hESC by specific factors as well as individual analysis of these. In the first study, hESC are sequentially differentiated first by Activin A (AA)-induction towards DE, and then by treatment with fibroblast growth factor 4 (FGF4) and retinoic acid (RA) to induce differentiation towards foregut endoderm. Importantly, FGF4 does not act as a posterior morphogen as can be observed in vivo, but rather plays an important role in cell survival. However, in combination with RA, which is known to induce dorsal pancreatic fates in vivo, a high level of PDX1+ posterior foregut endodermal cells, characterized by coexpression with SOX9, HNF6, and FOXA2, is obtained. In another study, we demonstrate a novel role of FGF2 as a broad patterning agent in hESC-differentiation. Low levels promote a hepatic fate, intermediate levels promote a pancreatic fate, whereas high levels promote lung and small intestinal fates. Importantly, with this protocol, a fraction of the PDX1+ cells were also positive for NKX6.1, indicating a putative pancreatic progenitor fate. HESC differentiation is most often followed by analyzing gene and protein expression, whereas functional assays to fully understand the in vivo potential of the derived cell types are rare. Here, we present the establishment of a functional in vivo assay in chick embryos for putative DE. The putative DE is derived by directed differentiation from mouse and human ESCs. Putative mouse and human DE is inserted in early chick embryos and integration into the chick endoderm is assayed 48 h later. Specifically, this thesis summarize the outcome of two important differentiation protocols that have provided knowledge on the individual roles of FGF4, RA and FGF2 in directing hESC towards a pancreatic fate, and provide a basis for further optimization towards obtaining pancreatic progenitors, endocrine progenitors, and ultimately fully functional β-cells for future cell replacement therapy.
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