UC Berkeley

Directed genetic reprogramming of cells from one identity to another offers tremendous potential in regenerative medicine, disease modelling and drug testing. However, its application is limited by the low efficiency at which it occurs, and existing methods to improve efficiency mostly utilize additional molecular biology and biochemical manipulations. This thesis explored an alternative paradigm for improving reprogramming efficiency: presentation of physical cues. To this end, I first showed that simply agitating an adherent culture with an orbital shaker enhanced its efficiency of reprogramming to induced pluripotent stem cells (iPSCs). I further demonstrated that convective mixing of the culture medium by orbital agitation blunted the upregulation of CDK inhibitor p57/Kip2 that was caused by the culture becoming overconfluent, which in turn enhanced the efficiency of reprogramming to iPSCs. Next, I showed that culturing reprogramming cells on solid supports scored with microgrooves enhanced their reprogramming into cardiomyocytes. I demonstrated that the microgrooves caused upregulation of the activity of the transcription factor megakaryoblastic leukemia-1 (Mkl1) / myocardin-related transcription factor A (Mrtf-a) and also enhanced organization of sarcomeric structure, with both effects contributing to better reprogramming efficiency. In addition to physical cues, I also explored whether treatment with only small molecules could reprogram fibroblasts into skeletal muscle cells. Indeed, I found that an optimized basal medium (10% FBS in DMEM with 50 μg/ml of ascorbic acid and 50 ng/ml of basic fibroblast growth factor (bFGF)) containing just 2 small molecules— 616452 [an inhibitor of the protein kinase activity of the transforming growth factor-beta (TGF-β) type I receptor (R1)] and forskolin (a plant diterpene that stimulates adenylyl cyclase and elevates the intracellular level of 3',5'-cyclic-AMP)—was sufficient to achieve reprogramming at high efficiency. In summary, this thesis described how both physical and chemical cues can contribute to enhancing the reprogramming of cell identity.