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

Abstract

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.

Microencapsulated Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells: Optimizing 3D Culture for Tissue Engineering Applications

“…To optimize the homogeneity of the resulting population, we chose to encapsulate the cells at the iHB stage. Unexpectedly, and unlike the encapsulation of hESC-derived DE described by Xie et al [ 37 ], iHBs encapsulated as single cells displayed a very low viability rate. Therefore, the alginate encapsulation of iHBs as single cells failed to provide a microenvironment that supported cell viability.…”

“…Several research groups have encapsulated pluripotent stem cells and investigated hepatocyte differentiation, though their analysis of liver functions has been limited [ 34 , 35 , 36 , 37 ]. Recently, Syanda et al, evaluated the encapsulation of hESC-derived hepatocyte aggregates in modified alginate and demonstrated the advantages of using this approach for transplantation in a mouse model [ 26 ].…”

“…Xie et al, compared the encapsulation stages of hESCs in order to optimize their hepatic differentiation in alginate beads. They showed that encapsulating hESC-derived definitive endoderm (DE) was more effective than encapsulating hESCs in terms of the degree of differentiation and viability of the hepatocytes produced in alginate [ 37 ].…”

“…Although HepaRG TM exhibit the most similar liver functions to PHHs, the hepatic activities of our E-iHep-Orgs were more substantial (see Table S5 ) and thus closer to PHHs. Xie et al, outlined that although it was promising, their approach was not efficient enough to generate a homogeneous and fully mature hepatocyte population [ 37 ]. Their encapsulated derived hepatocytes still expressed AFP, a marker of immature hepatocytes.…”

Design of Functional RGD Peptide-Based Biomaterials for Tissue Engineering

“…Furthermore, this technique, as well as other tissue-repairing techniques, may be underlined by complex mechanisms that require more exploration [ 69 , 70 , 71 ]. A three-dimensional culture of stem cells in media supplemented with an RGD-alginate gel increased cell viability, whereas, for a stem cell-derived embryonic body, alginate without RGD caused dissociation and an abnormal cyclic structure [ 73 , 74 ]. The formation of the retinal pigmented epithelium (RPE) from stem cells in the presence of scaffolds containing RGD alginate has been investigated [ 75 ].…”