An important advantage of employing extracellular matrix (ECM)-derived biomaterials in tissue engineering is the ability to tailor the biochemical and biophysical microenvironment of the cells. This study aims to assess whether three-dimensional (3D) liver-derived ECM hydrogel (LEMgel) promotes physiological function of liver organoids generated by self-organization of human hepatocarcinoma cells together with human mesenchymal and endothelial cells. We have optimized the decellularization method to fabricate liver ECM derived from sheep to preserve the greatest content of glycosaminoglycans, collagen, laminin, and fibronectin in produced LEMgel. During gelation, complex viscoelasticity modulus of the LEMgel (3 mg/mL) increased from 186.7 to 1570.5 Pa and Tan Delta decreased from 0.27 to 0.18. Scanning electron microscopy (SEM) determined that the LEMgel had a pore size of 382 ± 71 µm. Hepatocarcinoma cells in the self-organized liver organoids in 3D LEMgel (LEMgel organoids) showed an epithelial phenotype and expressed ALB, CYP3A4, E-cadherin, and ASGPR. The LEMgel organoid had significant upregulation of transcripts of ALB, CYP3A4, CYP3A7, and TAT as well as downregulation of AFP compared to collagen type I- and hydrogel-free-organoids or organoids in solubilized LEM and 2D culture of hepatocarcinoma cells. Generated 3D LEMgel organoids had significantly more ALB and AAT secretion, urea production, CYP3A4 enzyme activity, and inducibility. In conclusion, 3D LEMgel enhanced the functional activity of self-organized liver organoids compared to traditional 2D, 3D, and collagen gel cultures. Our novel 3D LEMgel organoid could potentially be used in liver tissue engineering, drug discovery, toxicology studies, or bio-artificial liver fabrication.

The availability of disease-specific induced pluripotent stem cells (iPSCs) offers a unique opportunity for studying and modeling the effects of specific gene defects on human liver development in vitro and for testing small molecules or other potential therapies for relevant liver disorders. Here we report, for the first time, the derivation of iPSCs by the retroviral transduction of Yamanaka's factors in serum and feeder-free culture conditions from liver-specific patients with tyrosinemia, glycogen storage disease, progressive familial hereditary cholestasis, and two siblings with Crigler-Najjar syndrome. Furthermore, they were differentiated into functional hepatocyte-like cells efficiently. These iPSCs possessed properties of human embryonic stem cells (hESCs) and were successfully differentiated into three lineages that resembled hESC morphology, passaging, surface and pluripotency markers, normal karyotype, DNA methylation, and differentiation. The hepatic lineage-directed differentiation showed that the iPSC-derived hepatic cells expressed hepatocyte-specific markers. Their functionality was confirmed by glycogen and lipid storage activity, secretion of albumin, alpha-fetoprotein, and urea, CYP450 metabolic activity, as well as LDL and indocyanin green uptake. Our results provide proof of principal that human liver-disease specific iPSCs present an exciting potential venue toward cell-based therapeutics, drug metabolism, human liver development and disease models for liver failure disorders.

Recent advances in human embryonic and induced pluripotent stem cell-based therapies in animal models of hepatic failure have led to an increased appreciation of the need to translate the proof-of-principle concepts into more practical and feasible protocols for scale up and manufacturing of functional hepatocytes. In this study, we describe a scalable stirred-suspension bioreactor culture of functional hepatocyte-like cells (HLCs) from the human pluripotent stem cells (hPSCs). To promote the initial differentiation of hPSCs in a carrier-free suspension stirred bioreactor into definitive endoderm, we used rapamycin for "priming" phase and activin A for induction. The cells were further differentiated into HLCs in the same system. HLCs were characterized and then purified based on their physiological function, the uptake of DiI-acetylated low-density lipoprotein (LDL) by flow cytometry without genetic manipulation or antibody labeling. The sorted cells were transplanted into the spleens of mice with acute liver injury from carbon tetrachloride. The differentiated HLCs had multiple features of primary hepatocytes, for example, the expression patterns of liver-specific marker genes, albumin secretion, urea production, collagen synthesis, indocyanin green and LDL uptake, glycogen storage, and inducible cytochrome P450 activity. They increased the survival rate, engrafted successfully into the liver, and continued to present hepatic function (i.e., albumin secretion after implantation). This amenable scaling up and outlined enrichment strategy provides a new platform for generating functional HLCs. This integrated approach may facilitate biomedical applications of the hPSC-derived hepatocytes.