TRANSCRIPTOME ANALYSIS OF HEPATOCYTES DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM CELLS

Background Human hepatocytes, within liver in vivo milieu, are capable of proliferation, but such characteristic is not reproducible in vitro when they were cultured over extended periods. In this scenario, induced pluripotent stem cells (iPS) arise as a promising source of hepatocytes, however, the differentiation outcome is still a challenge. In this study hepatocytes (iHep) differentiated from human iPS (hiPS) cells were characterized to evaluate the hepatic phenotype. Methods The hiPS cells differentiation protocol was carried out using RPMI medium supplemented from day 1 (D1) until D2 with B27- insulin (1%), Activin-A (100 ng/mL) and CHIR99021 (3 uM), from D3 to D5 the CHIR was removed. From D5 to D10, B27 (1%) and HGF (20 ng/mL) were added, and at D11 the HGF was replaced by SB431542 (1 uM) until the end of protocol, at D28. To evaluate differentiation\u0027s efficacy the expression and presence of hepatic markers were performed. Albumin (ALB), alfa-fetoprotein (AFP), hepatocyte nuclear factor4 (HNF4), CYP2D6, ASGR1 and Transferrin by qPCR, ALB and HNF4 by immunofluorescence, and ALB secretion by ELISA. Data were analyzed by Student\u0027s t test for paired variables and differences were considered significant at p Results The iHep cells expressed all hepatic genes evaluated, by qPCR, similar to HepG2. iHep also presented hepatic proteins (ALB and HNF4), detected by immunofluorescence. ELISA assay showed that iHep secreted ALB (96 ng/mL). As expected, HepG2 secreted ALB (263 ng/mL) and no ALB detection were observed in hiPS. The transcriptome analysis showed that iHep cells expressed hepatocyte-specific transcripts which were not found in hiPS. Conclusion The differentiation protocol from hiPS to iHep showed efficacy in generating hepatocyte-like cells and transcriptome analysis showed high genes similarity between iHep cells compared to human hepatocytes.