Engraftment Potential of Spheroid-Forming Hepatic Endoderm Derived from Human Embryonic Stem Cells

Abstract

Transplantation and drug discovery programs for liver diseases are hampered by the shortage of donor tissue. While recent studies have shown that hepatic cells can be derived from human embryonic stem cells (hESCs), few cases have shown selective enrichment of hESC-derived hepatocytes and their integration into host liver tissues. Here we demonstrate that the dissociation and reaggregation procedure after an endodermal differentiation of hESC produces spheroids mainly consisted of cells showing hepatic phenotypes in vitro and in vivo. A combined treatment with Wnt3a and bone morphogenic protein 4 efficiently differentiated hESCs into definitive endoderm in an adherent culture. Dissociation followed by reaggregation of these cells in a nonadherent condition lead to the isolation of spheroid-forming cells that preferentially expressed early hepatic markers from the adherent cell population. Further differentiation of these spheroid cells in the presence of the hepatocyte growth factor, oncostatin M, and dexamethasone produced a highly enriched population of cells exhibiting characteristics of early hepatocytes, including glycogen storage, indocyanine green uptake, and synthesis of urea and albumin. Furthermore, we show that grafted spheroid cells express hepatic features and attenuate the serum aspartate aminotransferase level in a model of acute liver injury. These data suggest that hepatic progenitor cells can be enriched by the spheroid formation of differentiating hESCs and that these cells have engraftment potential to replace damaged liver tissues.

Sustained Delivery Growth Factors with Polyethyleneimine‐Modified Nanoparticles Promote Embryonic Stem Cells Differentiation and Liver Regeneration

“…In our study, we employed day‐3‐induced DE cells as a source of isolated cells for transplantation into injured liver. The results showed DE cells were able to differentiate into hepatocyte‐like cells and attenuate AST and ALT levels after transplantation into injured liver, which confirmed the results of related reports 55, 56, 57. To our knowledge, this is the first report demonstrating that PEI‐MSNs can be used as a delivery system for growth factors to improve DE cells from mESCs in vivo and significantly restore liver function.…”

Engrafted human stem cell–derived hepatocytes establish an infectious HCV murine model

“…Recently, coculture of hiPSC-derived hepatocytes with human umbilical vein endothelial cells and human mesenchymal stem cells has been described to result in formation of 3D "liver buds" in vitro that could be efficiently engrafted and vascularized (53). Engraftment of spheroid-forming HLCs into CCl 4 -injured mice has also been described (54). Finally, two studies showed that human induced hepatocytes, transdifferentiated from human fibroblasts, could be engrafted in the liver parenchyma of transgenic mice and repopulate up to 0.3% to 4 Three months pi, all the inoculated HLC-engrafted mice showed high serum titers of HCV RNA (4-6 log [copies HCV RNA/ml]).…”

Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition

“…Previous studies have shown that primary hepatocytes and hiPSC-derived HLCs grown in 3D culture retain their hepatic features better when compared to their counterparts in 2D culture 74 75 76 77 78 79 80 81 . Most of the published protocols for the hepatic differentiation of hiPSCs or hESCs into HLCs have paired 2D culture during early stage of differentiation with subsequent 3D culture to promote assembly of differentiated cells for final maturation 32 78 82 83 84 85 86 . Our differentiation protocol was performed completely in 3D culture, using a new ROCKi-free and spin-free technique for EB formation 29 33 .…”