000164021 001__ 164021
000164021 005__ 20190509132358.0
000164021 0247_ $$2doi$$a10.5075/epfl-thesis-5027
000164021 02470 $$2urn$$aurn:nbn:ch:bel-epfl-thesis5027-6
000164021 02471 $$2nebis$$a6341385
000164021 037__ $$aTHESIS
000164021 041__ $$aeng
000164021 088__ $$a5027
000164021 245__ $$aDefinition of in Vitro Microenvironments to Characterize and Control Pancreatic Progenitor Expansion and Differentiation
000164021 269__ $$a2011
000164021 260__ $$bEPFL$$c2011$$aLausanne
000164021 300__ $$a130
000164021 336__ $$aTheses
000164021 520__ $$aThe pancreas plays a central role in metabolism. The  exocrine pancreas, composed of ductal and acinar cells,  secretes and delivers digestive enzymes into the duodenum  where they contribute to food digestion. The endocrine  pancreas, constituted by the islets of Langerhans, secretes  hormones in the blood to control glucose levels. In  particular, insulin is the hormone produced by β-cells  that instructs the peripheral tissues to uptake circulating  glucose. Diabetes mellitus is a heterogeneous disease characterized  by deregulated glucose homeostasis due to an impaired insulin  function. The advancements in clinical practice have made of  diabetes a chronic, instead of a lethal, disease; yet no  definitive cure is available. Beta-cell transplantation  offers promising results, especially for type 1 diabetes  where β-cells are selectively eliminated by an  autoimmune attack. Unfortunately its application suffers from  the scarcity of transplantable pancreas/islets from cadaveric  donors. Human embryonic stem cells (ESCs) or induced  pluripotent stem cells (iPSCs) would constitute unlimited  sources of transplantable β-cells, but the available  differentiation protocols are not yet optimal, especially  with regards to the numbers and the functionality of the  generated β-cells. It is anticipated that a stepwise  protocol would succeed in generating mature β-cells  in vitro if it is capable of fully mimicking the  embryonic development of these cells. Many aspects of pancreatic organogenesis have been  elucidated and could serve as a guide for driving the  commitment to β-cells. Unfortunately, pancreatic  progenitors in vitro do not behave as in the intricate  context of an embryo. This severely limits both the  understanding of pancreatic development at the single-cell  level and our ability in manipulating the process. The aim of our work was to develop in vitro  methodologies to sustain pancreatic progenitor culture and  expansion and to establish conditions to manipulate their  progeny. To this aim, we combined an informed approach based  on developmental biology and an empirical one. We first  observed that pancreatic epithelial explants from embryonic  day 10.5 mice could be cultured in presence of  Matrigel™ and exogenous FGFs even in the absence of the  mesenchyme that normally surrounds the epithelium. Notably,  the removal of mesenchyme did not affect the endocrine  commitment pattern of pancreatic progenitors. We then defined culture conditions allowing for the  expansion of dissociated embryonic pancreatic progenitors in  a three-dimensional Matrigel™-based environment. Under  these conditions, progenitors proliferated and self-organized  to generate pancreatic organoids containing both progenitors  and differentiated cells, mostly exocrine, after 7 days of  culture. When grafted into recipient pancreatic explants, the  cultured progenitors contributed to the three epithelial  pancreatic lineages (ductal, endocrine, acinar), integrating  seamlessly into the endogenous cellular network. Thus,  cultured progenitors retained their potential to  differentiate into endocrine cells, but expressed it only in  the appropriate niche. Subsequent removal of single  components from the culture system led to the identification  of some essential requirements for the maintenance/expansion  of dissociated pancreatic progenitors, such as a strong FGF  signaling and the Rho-associated kinase (ROCK) inhibitor  Y-27632. In addition to this, we observed that only small  clusters of pancreatic progenitors, but not single cells,  were able to generate pancreatic organoids, suggesting a  pivotal role for intercellular signaling in progenitor  maintenance and expansion. By manipulating the components of the medium, we could  affect fate commitment. In particular, the removal of FGF1  increased the yield of endocrine cells generated in  vitro. In our search for a full control over the in  vitro niche, we explored chemically defined matrices to  replace Matrigel™. We showed that pancreatic  progenitors could be cultured on laminin-functionalized  hydrogels, although less efficiently than in Matrigel™.  Moreover, differentiation into exocrine and endocrine cells  occurred spontaneously under these conditions. By comparing Matrigel™, hydrogel microwells and  two-dimensional culture systems, we uncovered the importance  of a tridimensional architecture for pancreatic progenitor  maintenance. We showed that pancreatic progenitors lost their  identity as they flattened onto 2D surfaces, whereas 3D  culture systems maintained the epithelial character of  pancreatic progenitors and allowed the acquisition of apical  polarization. To conclude, our work provides the first detailed  characterization of in vitro culture systems for  expansion and manipulation of dissociated embryonic  pancreatic progenitors. Further implementation will hopefully  allow the establishment of a fully-defined in vitro  niche for developing pancreas with potential fundamental  implications for the expansion of ES-derived pancreatic  progenitors and their differentiation into functional  β-cells.
000164021 6531_ $$apancreas
000164021 6531_ $$adevelopment
000164021 6531_ $$a3D in vitro culture
000164021 6531_ $$aMatrigel™
000164021 6531_ $$aPEG-hydrogel
000164021 6531_ $$alaminin
000164021 6531_ $$a[beta]-cell
000164021 6531_ $$aPdx1
000164021 700__ $$0243265$$g176580$$aGreggio, Chiara
000164021 720_2 $$aGrapin-Botton, Anne$$edir.$$g169494$$0240727
000164021 8564_ $$uhttps://infoscience.epfl.ch/record/164021/files/EPFL_TH5027.pdf$$zTexte intégral / Full text$$s17932711$$yTexte intégral / Full text
000164021 909C0 $$xU11258$$0252167$$pUPGRA
000164021 909CO $$pthesis-bn2018$$pDOI$$ooai:infoscience.tind.io:164021$$qDOI2$$qGLOBAL_SET$$pthesis
000164021 918__ $$dEDMS$$cISREC$$aSV
000164021 919__ $$aUPGRA
000164021 920__ $$b2011
000164021 970__ $$a5027/THESES
000164021 973__ $$sPUBLISHED$$aEPFL
000164021 980__ $$aTHESIS