*Purpose: Mouse pluripotent stem cell (PSC)-derived endoderm precursors (EPs) have small size, endoderm lineage gene expression signatures, and high proliferation rates, that allow engraftment in quiescent liver and create significant biomass without injury to the liver. This holds promise for the treatment of critical metabolic errors, where the structurally normal liver cannot tolerate reduction of hepatic function, however the analogue in human cell-based systems is not fully developed. Here we demonstrate amalgamated culture/selection conditions for derivation human EPs with potential clinical utility.

*Methods: We achieve precise and reproducible maintenance of pluripotency and differentiation over a time course spanning 3 days (EP), 6/7 days (hepatic progenitor (HP)), and 19 day hepatocyte-like cells. RT-qPCR and western blotting measure lineage-specific biomarkers at the population-level, while high throughput quantitative microscopy and flow cytometry measure these on a per cell basis. Cell proliferation states and viability are assessed using the trypan blue exclusion assay, and short-term engraftment into the murine liver is detected by in vivo imaging with the IVIS system.

*Results: Both EP and HP cells can be generated at greater than 90% efficiency from human pluripotent stem cells, and these cells proceed to become HLCs in vitro. Additionally, day 3 EP cells and day 6 HP cells maintain a high proliferation rate (30h and 22h, respectively). However, in contrast, day 6 HP cells are significantly larger than day 3 EPs. Early transplant analyses indicate EP cells can engraft in the undamaged murine liver parenchyma and persist for 8 days (see figure), however observation of HP-derived cells at this timepoint is 1/3 partial hepatectomy dependent. Additionally a high rate of mortality was observed with HP transplants, which may be due to their larger size leading to portal thrombosis. The high efficiency of EP differentiation and evidence of persistence in the undamaged mouse liver suggests these cells may be superior transplant candidates.

*Conclusions: We find human EPs recapitulate many of the desirable characteristics we have identified using mouse EPs, which can engraft and persist long-term in the undamaged quiescent liver. Pending longer term analysis of in vivo engraftment outcomes in appropriate models, these defined cell types are nearing the threshold of clinical testing as therapeutic agents for critical inborn errors of metabolism such as urea cycle defects.

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