Generating A Non-integrating Human Induced Pluripotent Stem Cell Bank From Urine-derived Cells

Human somatic cells can be reprogrammed back into pluripotent state as induced pluripotent stem cells that can undergo unlimited self-renewal while maintaining the potential to differentiate into all three germ layers. Human iPS cells hold great potential to be used for personalized regenerative medicine, drug discovery and disease modeling, for circumventing immunological response and ethical issues. To date, human iPS cells had been successfully generated from different tissue sources, such as skin fibroblasts and blood cells, although the reprogramming efficiencies varied much for different cell types. Moreover, the virus-based approach used in most of these reports raised the safety concerns, which hampered their further applications. To enable the application of iPS technology, it is essential to develop and optimize a practical approach to generate iPS cells under a feeder-free, virus-free and serum-free condition. Establishing a human iPS cell bank with diverse genetic backgrounds will facilitate the comprehensive study of iPS cells for disease modeling and mechanical analysis.Urine-derived cells are the most accessible somatic cell source for donation based on our anonymous survey. Our approach for isolating urine-derived cells can be universally accomplished by donors with broad age range and various genetic backgrounds. We have therefore collected50urine-derived cell lines from donors with age ranging from2to70years old, including patients with nervous system diseases or blood disorders.We established a method to generate human iPS cells from urine-derived cells under feeder-free, virus-free, serum-free condition. We used episome to deliver reprogramming factors in order to generate non-integrating iPS cells and confirmed the urine cell-derived iPS cells were free of exogenous transgenes and pluripotent. Since urine-derived cells were more susceptible to be reprogrammed, the high efficiency of reprogramming (0.015%) also allowed us to generate non-integrating iPS cells in a shorter time (20days). Moreover, fewer reprogramming factors were added compared to fibroblasts, oncogene c-MYC could be substituted by miR-302-367cluster to obtain safer iPS cells.The method of iPS cells generation from urine-derived cells described here was highly feasible. Using this approach, we have generated97iPS cell lines from20donors with diverse genetic backgrounds. These cells will be properly stocked after a set of standard characterization to form a human UC-iPS bank.We will further optimize our method at different steps including isolation and culture of urine-derived cells, electroporation and the culture condition of human iPS cells, so the whole process can be adapted to meet the requirements of cGMP, thus eventually facilitate the applications of iPS cells for both fundamental research and regenerative medicine.