Human cerebral organoids generated using urinary epithelial cell-derived induced pluripotent stem cells

Background: Human induced pluripotent stem cells (hiPSCs) provide a great promise for the success of novel disease model and regenerative medicine. Such remarkable cells may be derived using virtually any types of somatic cells through cell reprogramming and used for addressing questions relevant to the physiopathological development of human organs. The generation of hiPSCs using somatic cells that are obtained by noninvasive approaches would be ideal for the further development of hiPSC-based models to address childhood disease. Given the pluripotency in hiPSCs, we anticipate that hiPSCs generated from cells isolated in urine samples can be used for creating a three-dimensional organoid model system to recapitulate human brain development. Purpose: We desire the generation of transgene-free hiPSCs using urinary epithelial cells collected from the urine samples of health donors. We will further test cellular pluripotency and the capacity of forming three-dimensional cerebral organoids (minibrains) in these hiPSCs. Method: We use a polycistronic, self-replicating RNA system to express four transcription factors SOX2, KLF4, OCT4, and MYC in urinary epithelial cells that are isolated from the mid-to-late stream urine samples of healthy individuals. We also generate cerebral organoids from urinary epithelial cells-derived hiPSCs and other bona fide human pluripotent stem cell (hPSC) lines using an established differentiation protocol. Results: Urinary epithelial cells were successfully collected from four individuals, cultured, and reprogrammed into hiPSCs. Reprogrammed urinary epithelial cells express pluripotency markers and form cell types showing biomarkers for all three germ layers in undirected differentiation. The bona fide hPSCs have been used to optimize the procedure of building three-dimensional cerebral organoids. After matrigel embedding, the hPSC spheres that were precommitted to neuronal differentiation further developed complex structures that resemble discrete but interdependent cerebral regions, including forebrain, hippocampus, cortex, hindbrain, and a neural stem cell zone. These organoids stain positive for TUBB3 (a biomarker for neurons), GFAP (a biomarker for astrocytes), and SOX2 (a biomarker for neural stem cell populations). Conclusions: Our preliminary data suggest that hPSCs can reproducibly form minibrains in a defined culture condition. We will use the optimized procedure to test the capacity of urinary epithelial cells-derived hiPSCs in forming three-dimensional cerebral organoids (minibrains) and further establish a suitable model for studying brain developmental abnormalities associated with childhood disease.