Studies On Induction Of Embryonic Stem Cells To Pancreas Endocrine Cells And The Molecular Mechanism

Diabetes is a devastating chronic disease afflicting hundreds of millions ofindividuals. Although modern medicine has made considerable progress in thetreatment of diabetes, the morbidity and increased mortality associated with diabeticcomplications are still the major concerns. Beta cell mass and function are decreased tovarying degrees in both type1and type2diabetes. Pancreas transplantation and islettransplantation emerged as effective treatments for patients with diabetes, but thesignificant shortage of donor organs remains an issue. Stem cells are self-renewing,clonogenic and multipotent cells having tremendous potential for the treatment ofseveral human diseases and potential source for regenerative medicine. In the future,the promise of stem-cell-derived islet cell replacement or regeneration therapy maythus offer therapeutic benefit to people with diabetes, but there are major challenges tobe overcome before clinical application.Several promising approaches to generate new β-cells have been developed inrecent years. These include directed differentiation of pluripotent cells such asembryonic stem (ES) cells. High-yield methods to differentiate cell populationsintodefinitive endoderm, pancreatic progenitors, and β-cells havebeen establishedusing growth factors and small molecules. However, the final step of directeddifferentiation to generate functional, mature β-cells in sufficient quantities is yet to beachieved in vitro. Beside the need of transplantation medicine, a renewable source ofβ-cells would also be important in terms of present topographical structures,architecture and mimicking the natural surroundings of islet and to seek for alternativetreatment, finally, generating functional, mature β-cells.Three-dimensional (3D) cell cultures that mimic in vivo microenvironmentscontribute to study tissue structure related to organ differentiation and function.several studies have proved that3D culture can promote stem cell differentiation, andrestore cell function. In this study, our first aim was to verify the relationship between3D culture and function of β-cell. The second aim was to study the effects of3Dculture on ES cell differentiation. Part1Three Dimension Culture enhance function of β cell line MIN6: In thisstudy, mouse insulinoma6(MIN6) cells were cultured in a rotated3D culture systemto form islet-like aggregates, and glucose stimulated insulin secretion (GSIS) has beentested accordingly. We investigated whether the RhoA/ROCK pathway was involvedin the effect of3D culture on MIN6cells. The results demonstrated that moreendocrine-specific genes expressed and GSIS increased in the MIN6cells under3Dculture condition than that in monolayer culture system. We found that RhoA/ROCKinactivation led to F-actin remodeling in MIN6cell aggregates and more insulinexocytosis. Meanwhile, we also found that the gap junction forming protein,connexin36(Cx36), was increased in MIN6cell aggregates and the monolayer cellswith RhoA/ROCK inactivation,and GSIS dramatically decreased when Cx36wasknockdown by siRNA and couldn’t be reversed by RhoA/ROCK inactivation. Inconclusion, the RhoA/ROCK signaling pathway was involved in the insulin releasethrough increasing Cx36gap junctions in3D culture condition.Part2Three Dimension Culture Promotes Pancreas Endocrine Specificationsfrom hESC: Stem cell-based tissue engineering is a promising technology in the effortto create functional tissues of choice. To establish an efficient approach for generatingpancreas endocrine cells (PEC) directly from human embryonic stem cells (hESC) andto study the effects of three-dimensional (3D) culture on hESC differentiation, wecultured hESC on ultra-low attachment plates or biomimetic metrigel. Celldifferentiation was evaluated by Q-PCR, microscopy and flow cytometry analysis witha variety of pancreas specific markers. Our data indicate that hESC differentiated on3D culture developed multicell aggregates similar to those islet like structure; and theefficiency of PEC generation on3D, as indicated by the expression of variousPEC-specific surface markers (INS, GCG, SST), and INS positive cell wasreproducibly increased (23.7%) over their2D counterparts(16%). Moveover, insulinproduction stimulated by glucose in3D culture was found to be significantly higherthan that in2D differentiation system. We further demonstrate that3D culture inhibitsthe activation of focal adhesion kinase (FAK), and selective inhibition of this kinase issufficient to induce early endocrine commitment based on increased expression ofNGN3, PDX1and ISL1. Additional studies inhibit one of FAK downstream regulatorROCK suggest that ROCK pathway can also induce early endocrine specification. Wepropose that inhibition of SFK/FAK signaling can promote endocrine specification by limiting activation of the TGF-β/Smad2/3pathway. Moreover, we show that inhibitionof FAK and/or ROCK signaling promtes Cx36expression, this result hints thatincreases the expression of Cx36may contribute insulin secreation of late endocrinecells. Finally, we assessed the competence of hESC–derived PEC in vivo. The serumlevels of glucose-regulated C-peptide and glucose-tolerance tests of mice indicated thatthe hESC–derived PEC are functional and sufficient to control levels of glucose inserum.In summary, the results presented here demonstrated that3D culture contribute toGSIS and gene expression of mouse β-cell line MIN6.3D culture also provides ascalable system to achieve hESC proliferation and differentiation into PEC, wherebythe efficiency of ESC differentiation increased within3D systems, in terms of insulinproductivity, in comparison with2D differentiation systems. The mechanism is viainhibion FAK and its downstream singling way ERK, ROCK and Smad2. Thisresearch not only propose a theory and metherd for ESC differentiate to PEC, but alsoprovide a compelling evidence that hESC may serve as a renewable source of islets fordiabetes cell-replacement therapies.