Purpose: Embryonic stem (ES) cells have been shown to have great value therapeutically due to their limitless capacity for self-renewal and proliferation, and their ability to differentiate into all major cell lineages. However, the precise phenotype of the engrafting cell within the heterogeneity of the transplanted population of ES derived cells is uncertain. We aimed to identify and purify ES cell-derived endodermal precursor cells (EPs) and to explore their capacity for liver repopulation in mice after in vitro expansion.
Methods: We used an mSox17 promoter-driven Td-Tomato (definitive endoderm) targeted mES cell line in our study. Our medium for endodermal induction consists of a 3:1 mixture of DMEM and Hams/F12 medium supplemented with 0.5X N2 and 0.5X B27 supplements, 10ng/ml insulin-like growth factor 1 (IGF1), 0.1% BSA, 2 mmol/L glutamax, 0.1 mmol/L 2-mercaptoethanol, and 100 ng/ml ActivinA. The mSox17 expressing cells were obtained by flowcytometry. To drive hepatic differentiation, the day-5 endodermal cells were re-plated and change to Hepatocyte Culture Media (HCM). This was originally a three-step protocol: 2 days HCM supplemented with 30 ng/ml FGF4 +and 20 ng/ml BMP2, then 2 days HCM supplemented with 20ng/ml HGF, followed by 4 days HCM supplemented with 20ng/ml HGF, 10 ng/ml OSM, and 0.1 uM Dex. Gene expression analysis was done by quantitative PCR. Functional analysis associated with mature hepatocytes includes albumin secretion, glycogen storage, indocyanine green, low-density lipoprotein uptake, and inducible cytochrome P450 activity.
Results: Our endodermal induction method yielded a high percentage of mSox17 positive cells (up to 70%). After cell sorting based on Tdtomato, a homogeneous population of mSox17 expressing cells with 100% purity was achieved. The endoderm-like cells which we call endodermal precursors cells (EPs) were positive for FoxA2, Sox17, and AFP and could be further differentiated into hepatocyte-like cells by using our protocol, demonstrating hepatic morphology, functionality, and gene and protein expression. In Vivo data showed that the EPs are more capable of engraftment after liver transplantation in mice.
Conclusions: Our work provides insights toward the isolation of human pluripotent stem cell-derived endodermal precursor cells for potential therapeutic applications.
To cite this abstract in AMA style:Liu N, Fair J. Endodermal Precursor Cells Derived from Embryonic Stem Cells Can Engraft in Mouse Liver [abstract]. Am J Transplant. 2013; 13 (suppl 5). https://atcmeetingabstracts.com/abstract/endodermal-precursor-cells-derived-from-embryonic-stem-cells-can-engraft-in-mouse-liver/. Accessed May 7, 2021.
« Back to 2013 American Transplant Congress