STRATEGIES FOR HEPATOCYTE DIFFERENTIATION DERIVED FROM INDUCED PLURIPOTENT STEM CELLS USING SPHEROIDS

Background/Objective One of the main cells in the liver is the hepatocyte, which regulates the metabolism of various nutrients and xenobiotics, mediates endocrine function and excretion of toxic substances. Hepatocytes function in vitro have been studied for many years using primary hepatocytes and immortalized cells. Nevertheless, these cells show phenotypic instability or loss of adult liver functions and altered expression of important transcription factors. As an alternative to obtain a stable genetic background with preserved function and in large quantities, hepatocytes are being generated through differentiation of induced pluripotent stem cells (iPSC). However, one of the main problems to be overcome is the phenotype maturation. In this context, the objective of this work was to compare two different hepatocyte differentiation protocols using spheroids derived from iPSC. Methods Immunofluorescence for OCT3/4 and Nanog was performed to characterized pluripotent phenotype of iPSC and hepatocyte differentiation was induced for 28 days (D28). Two plating strategies were adopted to form the definitive endoderm: iPSC-monolayers (M) and iPSC-spheroids (S). To induce definitive endoderm, activin A and CHIR99021 were added to both plating conditions for 5 days. Prior hepatic maturation, iPSC-monolayers were also aggregated to form spheroids (MS). Subsequently, HGF and SB431542 were added to MS and S medium for 23 days. Spheroids were evaluated by real time PCR and immunofluorescence for hepatocyte-specific markers at D28. Results At protein level, the iPSC showed a pluripotent character through the nuclear presence of OCT3/4 and NANOG. At the end of the differentiation protocol, S differentiation strategy exhibited contractile cells. Albumin and cardiac troponin T were detected into the same spheroid by immunofluorescence. The MS strategy showed no beating cells. Also, CYP3A4 and albumin were detected by immunofluorescence and detectable mRNA levels were expressed for HNF4α, transferrin and alpha-1 antitrypsin. Conclusions Hepatocyte differentiation using spheroids requires a monolayer endoderm formation step in order to avoid the presence contractile cells at the end of protocol. Moreover, spheroid cells exhibited a great mature hepatocyte phenotype and can be used as a powerful tool for cell therapy, liver bioengineering, studies of drug efficacy and toxicity assays.