Comparing the morphology of iHeps and primary hepatocytes
We compared the differentiation of two GMP-compliant human pluripotent stem cell lines, one an embryonic stem cell (ESC) line and one an induced pluripotent stem cell (iPSC) line with primary adult human hepatocytes. The morphology of the cells at key stages of hepatocyte differentiation is shown in figure 1A: day 1 (PSCs), day 7 (definitive endoderm), day 11 (hepatoblasts) and day 21 (iHeps). On day 1 the cultures were ~40% confluent, with colony-to-colony contact. By day 7 both PSC lines had reached >90% confluency and exhibited irregular cell shapes, consistent with endodermal morphology. On day 11 both PSC lines had acquired a mixture of irregular and polygonal cell shapes. By day 21 both PSC lines had more defined polygonal cell shapes, with prominent nuclei and the presence of cytoplasmic vesicles, consistent with earlier reports.
When comparing the morphology of iHeps and primary hepatocytes, they shared a similar polygonal shape and size. However, the vesicles were less frequent in iHeps than primary hepatocytes (Fig. 1B). We speculate that the vesicles are glycogenic stores, a characteristic of mature hepatocytes.
iHeps retain a hepatoblast phenotype rather than undergoing full maturation
Mature, primary hepatocytes have a variety of differentiated functions, including detoxification, metabolism and protein synthesis. We assessed several functional markers at different stages of iHep differentiation: day 1 (pluripotent stem cells; ESC and iPSC), day 7 (definitive endoderm; DE), day 11 (hepatoblasts; HB) and day 21 (iHeps). We also compared them to mature primary hepatocytes (Fig. 1B).
Cytochrome P450 enzymes are monooxygenases expressed by mature hepatocytes that are involved in a variety of drug metabolism pathways. We measured native cytochrome CYP3A4 and CYP1A2 activity in iHeps and primary hepatocytes, normalized to cell number (Fig. 1B). There was no significant difference in CYP3A4 and CYP1A2 activity between the iHeps and primary hepatocytes.
We next analyzed the levels of albumin, alpha-fetoprotein (AFP) and urea in a 24 h conditioned medium. Albumin functions as a modulator of plasma oncotic-pressure and to transport ligands, including drugs and bilirubin. In contrast to adult hepatocytes, iHeps did not produce a measurable amount of albumin (Fig. 1B). In healthy mature hepatocytes, ammonia is metabolized to urea; urea production was detected in hepatocyte but not iHep cultures (Fig. 1B). Finally we assessed supernatant levels of AFP, which is produced by hepatoblasts but not by mature hepatocytes. At day 11 of the differentiation protocol cells expressed high levels of AFP, indicative of successful hepatoblast differentiation. However, this was retained at day 21, indicating that the maturation had not been achieved (Fig. 1B).