Lineage Restriction and Differentiation of Human Embryonic Stem Cells into Hepatic Progenitors and Zone 1 Hepatocytes

Abstract

Human embryonic stem (hES) cells can self-renew, which enables them to have considerable expansion potential, and are pluripotent. If their differentiation can be controlled, they can offer promise for clinical programs in cell therapies. A novel strategy has been developed to derive early hepatocytic lineage stages from hES cells using four sequential inducing steps lasting 16 days. First, embryoid bodies (EBs) were generated by growing hES cells in suspension for 2 days; second, EBs were lineage restricted to definitive endoderm with 3 days of treatment with human activin A; third, cells were differentiated further by coculturing for 5 days with human fetal liver stromal cells (hFLSCs) made transgenic to stably release basic fibroblast growth factor (bFGF); fourth, treating them for 6 days with soluble signals comprised of hFLSC-derived bFGF, hepatocyte growth factor, oncostatin M, and dexamethasone. Induced cells displayed morphological, immunohistochemical, and biochemical characteristics of hepatocytic committed progenitors and of early lineage stage hepatocytes found in zone 1 of the liver acinus. They expressed alpha-fetoprotein, albumin, cytokeratin 18, glycogen, a fetal P450 isoform, and CYP1B1, and demonstrated indocyanine green uptake and excretion. In conclusion, we have developed a novel method to lineage restrict hES cells into early lineage stages of hepatocytic fates.

A Strategy Using Pluripotent Stem Cell-Derived Hepatocytes for Stem Cell-Based Therapies

“…Put simply, FGF produced from cardiac mesoderm triggers hepatic buds from the endoderm-originated tube, BMP-4 from septum transversum promotes differentiation and proliferation, cytokine, such as OSM, leads them to the final stage (Duncan, 2003). Many methods to differentiate have been developed and practiced (Cai et al, 2007;Hu et al, 2003;Ishii et al, 2008;Pei et al, 2009;Soto-Gutierrez et al, 2006;Teratani et al, 2005). Curiously, however, they deleted BMP-4 produced from septum transversum, which is an indispensible factor for hepatic development.…”

Preliminary characteristic of alginate, heparin-chitosan-alginate, heparin microencapsulated hepatocytes system

“…All procedures were performed under general (ether) anesthesia using sterile surgical techniques. Hepatocytes were harvested using an in situ two-step ethylenediaminetetraacetic acid/collagenase digestion, as described previously [10] . Isolated hepatocytes with a viability of more than 90% were used for the experiments.…”

Toward engineering of vascularized three-dimensional liver tissue equivalents possessing a clinically significant mass

“…Pei et al prepared genetically modified FGF-secreting human fetal stromal cells and applied them to human ES cells in the presence of HGF and OSM. They succeeded in obtaining well-defined hepatocyte progenitors and an early stage of hepatocytes [90].…”

“…Interestingly, they compared two kinds of such 3D pellets, that containing cells alone and cells with small intestinal submucosa (SIS) matrix, and observed hepatic differentiation was much superior in the pellets without SIS matrix. The results seem to suggest that contact with the liver-specific ECM or cells [84,[87][88][89][90] is favorable for hepatic induction of stem cells. Kazemnejad et al also reported hepatic differentiation of human MSCs in 3D culture using poly--caprolactone-based nanofiber scaffolds [98].…”