A principle obstacle in regenerative medicine is the lack of renewable sources of human cells for the transplant-based treatment of liver diseases and diabetes. Human biliary tree stem/progenitor cells (hBTSCs) have multipotential differentiation capability, comprising hepatocyte, cholangiocyte and pancreatic islet fates. Recently, we demonstrated that in human duodenum, submucosal glands (SG) contain cells with endodermal features and capable to differentiate in vitro and in vivo to liver and pancreatic islet fates. The translation of these discoveries to a next application requires pre-clinical studies. Adipose-derived stem cells (ASCs) are one of the most widely studied and used source in regenerative medicine, since they are endowed with favorable properties and release paracrine factors know to act positively in the process of tissue regeneration. The main aim of this project is to investigate the role of hBTSCs and duodenal SGs in rescuing experimental murine models of liver injuries and type I diabetes, and to increase the proliferation and the effects of these endodermal-derived cells through the use of ASCs. The project will be subdivided in working packages (WPs) with specific objectives. WP#1 will optimize long term in vitro expansion and organoid formation of hBTSCs and duodenal SGs through co-culturing with ASCs. WP#2 will compare the effects of hBTSCs and duodenal SGs differentiated in hepatocytes in engrafting into the liver and rescuing experimental models of liver injuries in SCID mice. WP#3 will compare the effects of hBTSCs and duodenal SGs differentiated in ß cells transplanted in the fat pad in rescuing a stretozotocin-induced model of diabetes in SCID mice. WP#4 will assess the potential application of both cellular sources to regenerative medicine combining the favorable features and functions of the endodermal stem cell populations with the beneficial roles played by ASCs and their secreted proteins in an in vitro and in an vivo modelling.
Regenerative medicine is one of the most exciting and rapidly advancing areas of modern bio-medical sciences as it focuses on innovative approaches to repair or replace cells, tissues and organs. The mission of our project is to overcome one of the principle obstacle in regenerative medicine: the lack of renewable sources of human cells for the transplant-based treatment of liver diseases and diabetes (1, 2). The impact of this project may be absolutely groundbreaking considering the millions of affected subjects may benefit from an effective cell product for the regenerative medicine of liver and pancreas, comprising diabetes (3, 4). Our project has a unique multidisciplinary and translation approach since it conjugates two very promising approaches: i) the use of determined stem/progenitor cells isolated from adult humans which have the advantage to require only minimal manipulation with respect to reprogrammed cells (2), and are the physiological precursors of the mature cells of the liver and pancreas, the hBTSCs (5) and the duodenal SGs (6), ii) and the use of ASCs, which clearly demonstrated multiple beneficial effects due to immuno-modulatory, trophic, pro-angiogenetic properties and paracrine signaling mechanisms (7-10). Our proposal comprises several potential advantage with respect the state of the art. In particular issues concerning immunity may be alleviated. Indeed, our previous investigation indicated that hBTSCs express null or minimally HLA antigens and induce Fas-mediated apoptosis of activated T-cells (11). Moreover, a further advance with respect our previous demonstrations may result from the co-administration of hBTSCs or duodenal SG cells with ASCs derived from the same patient or the co-administration of hBTSCs or duodenal SG cells with ASC-associated secretome (7-10). Although stem cells are usually hypoimmunogenic, in type I DM, transplanted cells are challenged by adaptive immune responses such as local inflammation and rejection (4). In addition, once maturated into insulin producing ß-cells, graft cells will be attacked by persistent autoreactive T cells in patients with T1DM (4). Our proposal minimizes the need of immunosuppression since hBTSCs can protect themselves from the immune attack trough Fas-L expression, and since ASCs and even their secretome have very well established immunomodulatory properties. hBTSCs and duodenal SGs have the advantages of other adult stem cell sources over embryonic stem cells and reprogrammed induced pluripotent stem cells (iPSCs), like the minimal manipulation in vitro, the complete lack of tumorigenity risk, the lack of ethical issues, the use of cells physiologically implicated in the regeneration of the target organ. Moreover, duodenal SGs can be retrieved in standard endoscopic biopsies as we have described recently (6), so they are a rare source of potential autologous stem cells (6). Notably, our project targets also the crucial problem of adult stem cell sources: the limited number of cells necessary to have the critical mass need for organ repopulation and disease rescuing (2). We will face the problem by the well-established technique of the organoids (6) and by the innovative use of the ASCs or their secretome to modulate the growth of hBTSCs and duodenal SG cells both in vitro and in vivo (9,10). We will evaluate protocols for the formation of organoids starting from a small amount of tissue. Our proposal contains further multiple advancements in the actual scenario: multiple cells with different biological properties (possibly synergic) from same donor, the use of the same multipotent stem cells to target both liver and pancreatic diseases optimizing the use of resources, the use of sources largely available both from adult and fetal donors (2). We previously obtained results which demonstrate the ability of the proponents to create reproducibly in vitro long-term expanding organoids from the biliary tree and duodenal SGs (6, 12). These results demonstrated the feasibility of the techniques, which will be used in this project, but also showed a stable phenotype in vitro and a well-defined 3D structure of the organoids (6, 12). Through the established collaboration between the research units attempts of co-culturing of hBTSCs have been already successfully tested.
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