Urine Cell-Derived Hepatocyte-Like Cells As Potential Therapeutic Cell Transplants for Different Liver Diseases

Hepatocytes derived from human somatic cells would be useful in regenerative medicine, drug development and cell-based disease models. Several types of somatic cells have been reprogrammed to induced pluripotent cells (iPSCs) and then differentiated to hepatocyte-like cells (iHeps). However, the method for generating such cells from renal epithelial cells shed in human urine has not been described systematically. Thus, we tested whether these urine cell-derived iPS cells will show the ability to differentiate into iHeps and may have the potential to engraft in mouse liver. 250 – 500 ml of fresh human urine was collected from 3 different donors. After several washing steps, the cell pellet was cultivated in one well of a 12 well plate. The isolated epithelial cells were reprogrammed into iPS cells by using transgene-free methods, all delivering the pluripotency factors Oct3/4, Sox2, Klf4 and c-Myc. After characterization of stable iPS cell lines, we started a three-step protocol for hepatic differentiation. To evaluate hepatic status during differentiation process we analyzed 94 genes using qPCR, as well as flow cytometry analysis, immunocytochemistry and hepatocyte-specific functional assays. One million iHeps were transplanted via spleen into PiZ/Scid mice, a model for alpha 1-antitrypsin deficiency. 5 days after urine-cell cultivation, 4 – 6 cell colonies were observed which outgrew to a stable cell population within the next 2 weeks. Reprogramming of urine cells produced iPS cell lines that indicated typical stem cell features. The first step of differentiation induced a substantial morphological change, while 90% of the cells expressed the definitive endoderm marker Sox17, shown by qPCR and immunocytochemistry. At the final stage, flow cytometry analysis revealed 86% of Albumin and 29% of ASGPR positive cells. Genes which are responsible for cholesterol homeostasis, bile transport and detoxification processes are upregulated, e.g. farnesoid X receptor (FXR), bile sort export pump (BSEP; ABCB11) and constitutive androstane receptor (CAR), shown on mRNA level. The iPS-derived iHeps showed glycogen storage, urea and albumin production. Engraftment of iHeps in livers of PiZ/Scid was proofed by immunohistochemistry. The results indicate that urine cell-derived iPS cells can be efficiently induced to differentiate into iHeps. We found that our methods allowed the expression of liver specific functions, which will be beneficial as potential therapeutic cell transplants for various liver diseases.