Baptista, Emma Moran, Shay Soker Background: Human hepatocytes derived from somatic cells of individuals would be useful in developing cell-based disease models, drug development

and regenerative medicine. Although several types of somatic cells have been reprogrammed to induced pluripotent cells (iPSCs) and then differentiated to hepatocyte-like Adriamycin supplier cells (iHep), the method for generating such cells from renal epithelial cells shed in human urine has not been described systematically. Aim: Reprogram-ming human urinary epithelial cells to iPSCs and differentiating them to iHeps. Methods: Fresh human urine (250-500ml) was collected and the washed cell pellets were expanded in culture in a defined growth medium. The epithelial cells were reprogrammed

into iPSCs by using non-transgene integrating methods, such as delivering the pluripotency factor genes OCT3/4, SOX2, KLF4 and MYC by nucleofection of episomal (EBNA) plasmids or infection with recombinant Sendai viruses. After characterization of stable iPS cell lines, a 3-step differentiation toward hepatocytes was performed. At each step, the expression pattern of 141 genes was assessed by qRT PCR. Flow cytometry, immunocytochemistry and hepatocyte-specific functional assays were performed. Results: After 2 weeks of cultivation of urinary cells, 4-6 stable cell populations emerged. Both reprogramming strategies yielded iPSCs with characteristic features and normal karyotype. The first step selleck screening library of differentiation generated definitive endoderm cells, with 90% of the cells expressing the definitive endoderm marker Sox17, as shown by qRT PCR and immunocytochemistry. At the final stage, flow cytometry revealed that 86% and 29% of the cells were positive for human serum albumin and human asialoglycoprotein receptor, respectively.

The iHeps expressed mRNAs for nuclear receptors that regulate genes involved in cholesterol homeostasis, bile acid transport and detoxification, including farnesoid X receptor (FXR), and constitutive androstane receptor (CAR/ NR1l3), as well as the bile salt export pump (BSEP/ABCB11). The iHeps exhibited glycogen storage, and secreted urea and albumin Casein kinase 1 into the media. Conclusions: Urine cell-derived iPSCs can be reprogrammed and then efficiently differentiated to iHeps. Our methods allowed the expression of liver specific functions. Thus, urine is a readily available source for generating human hepatocyte-like cells that could be potentially useful for disease modeling, pharmacological development and regenerative medicine. Disclosures: The following people have nothing to disclose: Vanessa Sauer, Xia Wang, Krisztina Tar, Tatyana Tchaikowskaya, Yanfeng Li, Chandan Guha, Namita Roy-Chowdhury, Jayanta Roy-Chowdhury Microengineering human tissues on a chip remains an open challenge from both scientific and technological points of view.