Direct Differentiation of Human Embryonic Stem Cells to 3D Functional Hepatocyte-like Cells in Alginate Microencapsulation Sphere.

BACKGROUND: The lack of a stable source of hepatocytes is one of major limitations in hepatocyte transplantation and clinical applications of a bioartificial liver. Human embryonic stem cells (hESCs) with a high degree of self-renewal and totipotency are a potentially limitless source of a variety of cell lineages, including hepatocytes. Many techniques have been developed for effective differentiation of hESCs into functional hepatocyte-like cells. However, the application of hESC-derived hepatocyte-like cells (hESC-Heps) in the clinic has been constrained by the low yield of fully differentiated cells, small-scale culture, difficulties in harvesting, and immunologic graft rejection. To resolve these shortcomings, we developed a novel 3D differentiation system involving alginate-microencapsulated spheres to improve current hepatic differentiation, providing ready-to-use hESC-Heps. METHODS: In this study, we used alginate microencapsulation technology to differentiate human embryonic stem cells into hepatocyte-like cells (hESC-Heps). Hepatic markers of hESC-Heps were examined by qPCR and Western blotting, and hepatic functions of hESC-Heps were evaluated by indocyanine-green uptake and release, and ammonia removal. RESULTS: The maturity and hepatic functions of the hESC-Heps derived from this 3D system were better than those derived from 2D culture. Hepatocyte-enriched genes, such as HNF4α, AFP, and ALB, were expressed at higher levels in 3D hESC-Heps than in 2D hESC-Heps. 3D hESC-Heps could metabolize indocyanine green and had better capacity to scavenge ammonia. In addition, the 3D sodium alginate hydrogel microspheres could block viral entry into the microspheres, and thus protect hESC-Heps in 3D microspheres from viral infection. CONCLUSION: We developed a novel 3D differentiation system for differentiating hESCs into hepatocyte-like cells by using alginate microcapsules.

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells.

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4α nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

Characteristics of various forms of phosphorus and their relationships in the sediments of Haizi Lake, China.

The phosphorus (P) distribution in the sediments of Haizi Lake from the middle reach of the Yangtze River region, China, was investigated using a sequential chemical extraction procedure. P forms and concentrations of sediment samples taken at 25 sites over the whole lake were measured. The relationships between various forms of P in sediments and dissolved P in the overlying water were also discussed. Results showed that the concentrations of total P (TP) in the sediments ranged from 404 to 670 mg kg⁻¹, with an average of 503 mg kg⁻¹. The exchangeable P (Ex-P), Al-bound P (Al-P), Fe-bound P (Fe-P), occluded P (Oc-P), authigenic carbonate fluorapatite + biogenic apatite + CaCO3-associated P (ACa-P), detrital apatite + other inorganic P (De-P) and organic P (Or-P) accounted for, on average, 0.52, 0.04, 10.9, 32.0, 7.4, 20.1 and 29.0% of TP, respectively. Relevance analysis indicated that Oc-P, ACa-P and De-P, as the majority forms of inorganic P, were less correlated to others. The significant correlations between Ex-P, Al-P, Fe-P, Or-P and TP suggested the probability of reciprocal transformation. It was suggested that Ex-P, Al-P, Fe-P, Or-P and TP in the sediments might be released easily to the water interface, resulting in sustained lake eutrophication.