Reprogramming of Mouse Fibroblasts to iPS Cells in Stirred Suspension Bioreactors using Physical and Genetic Methods.

摘要

The clinical application of stem cells depends on the availability of pluripotent cells that are not restricted by ethical, immunological and technical considerations. The recent development of the derivation of induced pluripotent stem cells (iPSCs) from somatic cells is opening a new era in developmental biology. Having potential advantages in regenerative medicine, iPSCs are similar to their embryonic stem cell (ESCs) counterparts and possess all of the essential criteria such as pluripotency, self-renewal and potency. Despite major improvements in the methods of iPSC generation and expansion, the process still remains inefficient and poorly characterized.;This thesis describes the development of a novel approach for the derivation and expansion of iPSCs. Initially, a study conducted to evaluate the potential of using the stirred suspension bioreactor (SSB) system for large scale differentiation of murine ESCs into cardiomyocytes. Despite the fact that we could differentiate ESCs into cardiomyocytes, surprisingly, we found that the SSB suppressed differentiation, in favor of maintaining pluripotency. The effect was presumed to be due to effect of fluid shear stress (FSS) on the cells. Based on the findings that SSBs favor pluripotency over differentiation, we examined this environment for the long term expansion and maintenance of iPSCs in an undifferentiated state. Our results demonstrated that SSBs yield a 58-fold expansion of undifferentiated pluripotent iPSCs over 4 days. In vitro and in vivo characterization further confirmed the existence of fully functional and undifferentiated pluripotent iPSC aggregates following long term passaging in SSBs.;Subsequently, we developed a novel method for the efficient and expedited derivation of iPSCs in SSBs. We found that suspension bioreactors increase both the kinetics and efficiency of iPSC derivation and can provide a selective advantage to enhance cellular reprogramming, presumably through application of shear stress. The resulting suspension-derived iPSCs (SiPSCs) resembled ESCs in their in vitro and in vivo characteristics, such as teratoma and chimera formation and displayed germ line transmission competency. The findings presented in this thesis show that SSBs not only suppress differentiation but also provide a novel environment for the expansion and maintenance of iPSCs as well as their efficient derivation.