Hepatocytes generated from iPSCs of different germ-layers secreted liver proteins into blood.

Researchers have demonstrated that mature and immature liver cells generated from induced pluripotent stem cells (iPSCs) derived from multiple adult cell types are as effective as both embryonic stem cell (ESC)-derived hepatocytes and primary human hepatocytes at engrafting and functioning in the livers of experimental mice. The Johns Hopkins University School of Medicine team that carried out the studies say iPSC-derived cells were equivalent to the ESC-derived cells and primary hepatocytes in terms of their capacity to regenerate damaged livers and with respect to the levels of human-specific liver proteins they secreted into the animals’ bloodstreams.

Hepatocytes derived from different cell types retained some epigenetic memory of their germ-layer origins and showed noticeable differences in epigenetic profiles to those of primary human hepatocytes, but this didn’t seem to affect their function in vivo. The team reports its findings in Science Translational Medicine in a paper titled “In Vivo Liver Regeneration Potential of Human Induced Pluripotent Stem Cells from Diverse Origins.”

Propagating large numbers of adult human hepatocytes ex vivo for cell therapy is hampered by the limited proliferation potential of the cells and their rapid loss of function and viability, report assistant professor of oncology Yoon-Young Jang, M.D., at the Johns Hopkins Kimmel Cancer Center, and colleagues. Moreover, while there have been great advances in liver stem cell biology, hepatic stem cells are relatively rare, which makes their isolation and expansion as an alternative source of liver cells unsuitable at the large scale.

Conversely, in vitro differentiation of both human ESCs and iPSCs into cells of hepatic lineage has been achieved as an alternative option. What we don’t yet know, the authors point out, is whether human iPSC-derived hepatic cells retain functionality after engraftment, and if so, whether this holds true for cells at various stages of differentiation and derived from different adult tissues.

Dr. Jang’s team used a mouse model of liver injury to test the regenerative capabilities of human hepatic cells generated from human iPSCs derived from a range of adult tissues including skin, blood, and liver cells. The researchers evaluated hepatic cells at various stages of differentiation and compared them with liver cells generated from human ESCs, and primary liver cells.

Genome-wide methylation analysis of the iPSCs first confirmed that the cells had similar methylation patterns to those derived from human ESCs and were distinct from those of parental cells. Despite the similarities in epigenetic profiles between iPSC lines derived from the different germ layers, their methylation patterns did indicate that a level of tissue-specific epigenetic memory was preserved according to their origins.

Implanting both immature and mature iPSC-derived hepatic cells into mouse models of liver cirrhosis resulted in levels of cell engraftment that were similar to those achieved using embryonic ESC-derived hepatocytes and primary hepatocytes. Transferring about two million iPSC-derived differentiated liver cells resulted in 8–15% engraftment rate, compared with 11% for adult human liver cells. Transferring even more cells—up to 7 million—resulted in an even higher engraftment rate, of about 35%. “Regardless of their tissue of origin, human iPSC-derived hepatic cells displayed equivalent liver engraftment when compared with ESC-derived hepatic cells as well as primary hepatocyte cells,” the authors state.

Further analysis of the animals’ blood and livers confirmed that the human ESC- and iPSC-derived cells differentiated into mature and functional human hepatocyte-like cells, as evidenced by the production of human albumin and CYP2E1 cytochrome P450 proteins.

The researchers were also able to detect measurable concentrations of multiple human liver-specific proteins including albumin, transferrin, AAT, and fibrinogen, which were comparable to those obtained from mice that had been transplanted with human primary hepatocytes, and correlated with the liver engraftment efficiencies. 

Interestingly, the team didn’t find any evidence of tumor formation over the seven month duration of the study—which is equivalent to over 30 years of human life—regardless of the sources or stages of donor cells. Possible explanations for this promising finding could relate to either the relatively low dose of human cells transplanted, the intravenous transplantation route, or the high efficiency of directed hepatic differentiation, Dr. Jang and colleagues suggest.

“In summary, these studies demonstrate the in vivo liver regenerative potential of human iPSC-derived hepatic cells at different stages of differentiation, and suggest that epigenetic memory retained in human iPSCs does not influence directed hepatic differentiation.”

The authors admit that further studies are needed to determine whether the hepatic derivatives of patient-specific iPSCs can also functionally engraft in mouse liver and recapitulate disease features for the purpose of creating in vivo disease models. They also stress that it still remains to be seen whether for some human cell types, such as blood, for example, epigenetic memory would have a more marked impact on the efficiency of directed differentiation. If it does, then even this apparent hurdle could potentially aid the directed differentiation of specific tissue types that have traditionally represented a challenge.

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