Investigating human pancreas development in a dish

Efforts are underway to develop cell therapies for diabetes using endocrine cells derived in vitro from human pluripotent stem cells. However, protocols are hampered by a scarcity of knowledge on human pancreatic development and on how to model endocrine differentiation of pancreatic progenitors in vitro. In this thesis, gene expression, Fluorescence Activated Cell Sorting (FACS) and immunohistochemical analyses were used, along with other techniques, to provide systematic characterisation of pancreatic cell populations during ontogenesis. Human fetal pancreatic progenitor cells (hPPCs) were successfully isolated from pancreatic tissue of different embryonic and fetal stages. hPPCs were extensively cultivated in 3D MatrigelTM cultures in Expansion Medium (EM), and in Organogenesis Medium (OM), to assess their multipotent nature. Freshly isolated or expanded hPPCs were triggered to differentiate into endocrine cells; notably a significant increase of endocrine markers was observed at gene and protein levels, even after extensive expansion. 3D organoid cultures rely mostly on MatrigelTM, a hydrogel derived from mouse sarcoma which is not suitable for clinical application. To overcome this, hPPC cultures were established utilizing fully synthetic, functionalised hydrogels with defined physical properties and also by producing hydrogels from native extracellular matrix (ECM). Cell growth and differentiation potency were modulated by ECM properties, opening the possibility of directing cell fate by integrating soluble factors, matrix components and cell-cell interaction mechanisms. In conclusion, this thesis provides a comprehensive characterisation of human embryonic and fetal pancreas, and addresses human pancreatic progenitor potency in clinically relevant culture conditions