Expansion and Derivation of pluripotent stem cells in stirred suspension bioreactorRegenerative Medicine Bioprocesses Cell Culture
We are developing methods for expanding mouse and human pluripotent stem cells (i.e. embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) as aggregates in stirred suspension bioreactors. Bioreactors offer numerous advantages over conventional static culture systems, which lack control and have low cell yields. Bioreactors can be controlled; physiological levels of key parameters can be maintained, and large amounts of cells/tissue can be grown in a short time. Human intervention is minimized, so there is little batch variability. This makes bioreactors an essential step in the application of stem cells. Having demonstrated that the quality of bioreactor expanded cells was equal if not superior to conventional static expansion methods we next explored our ability to differentiate mouse ESCs into different tissue types in the bioreactor. In sharp contrast to static culture, where cell differentiation occurred efficiently, we observed that many cells remained pluripotent in the suspension culture environment and causing them to be refractory to tissue differentiation.
Using the mouse model system, we recently demonstrated that in the bioreactor iPSCs form 1000-fold more efficiently, and in half-time compared to static culture. The method is so efficient that we have begun to remove genes from the reprogramming process. Our current research is investigating how fluid shear promotes cellular reprogramming via mechanotransduction.
Osteoblast, Chondrocyte and Cardiomyocytes DifferentiationTissue Engineering Differentiation Cell-based therapy
We are developing new methods for differentiating mouse and human pluripotent stem cells (PSCs) into osteoblasts, chondrocytes and cardiomyocytes and transplanting them into animal injury models. When cells are affixed to the bottom of micro-drop cultures and treated directly with differentiation factors, some cells undergo apoptosis while others form small chondrocyte or osteoblast fated aggregates in suspension. We have also developed methods to differentiate ESCs within collagen gels. These constructs are sufficient to induce osteoblast formation in the absence of any growth factors: ESCs are simply removed from pluripotency maintenance factors, suspended in collagen gels and cultured for 15 days whereupon they form osteoblasts or chondrocytes in vitro. We have developed a mouse burr-hole fracture model in the proximal tibia, one that is stable and has a known geometry. Osteoblast differentiations derived of male PSCs incorporate into female bone based on Y-chromosome in situ hybridization histochemistry, uCT, and magnetic resonance imaging. Cartilage differentiations are able to form bone when transplanted into animals via a process resembling endochondral ossification.