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.

Human embryonic stem cell (hESC)-derived hepatocytes provide a promising unlimited resource for the treatment of liver disease. However, current protocols for the generation of mature and functional hepatocytes are inefficient. Therefore, in order to better differentiate and maintain the function of differentiating hESCs, we have hypothesized that hESCs undergo better differentiation into hepatocyte-like cells (HLCs) when induced on three-dimensional nanofibrillar surfaces. We have demonstrated that, during stepwise differentiation of induction, the markers of hepatic lineage expressed and finally lead to the generation of functional mature cells. In the presence of an ultraweb nanofiber, HLCs produced lower AFP, greater urea, glycogen storage, metabolic PROD activity, uptake of LDL and organic anion ICG, all of which are indicative of the differentiation of HLCs. These results show that topographically treated hESCs at the nano level have a distinct hepatic functionality profile which has implications for cell therapies.

Organ and tissue shortage are known as a crucially important public health problem as unfortunately a small percentage of patients receive transplants. In the context of emerging regenerative medicine, researchers are trying to regenerate and replace different organs and tissues such as the liver, heart, skin, and kidney. Liver tissue engineering (TE) enables us to reproduce and restore liver functions, fully or partially, which could be used in the treatment of acute or chronic liver disorders and/or generate an appropriate functional organ which can be transplanted or employed as an extracorporeal device. In this regard, a variety of techniques (e.g., fabrication technologies, cell-based technologies, microfluidic systems and, extracorporeal liver devices) could be applied in tissue engineering in liver regenerative medicine. Common TE techniques are based on allocating stem cell-derived hepatocyte-like cells or primary hepatocytes within a three-dimensional structure which leads to the improvement of their survival rate and functional phenotype. Taken together, new findings indicated that developing liver tissue engineering-based techniques could pave the way for better treatment of liver-related disorders. Herein, we summarized novel technologies used in liver regenerative medicine and their future applications in clinical settings.

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Dynamic suspension culture of human pluripotent stem cells (hPSCs) in stirred bioreactors provides a valuable scalable culture platform for integrated differentiation toward different lineages for potential research and therapeutic applications. However, current protocols for scalable and integrated differentiation of hPSCs limited due to high cost of growth factors and technical challenges. Here, hPSCs aggregates primed with 6 and 12 μM of CHIR99021 (CHIR), a Wnt agonist, in combination with different concentrations of high cost Activin A (10, 25, 50, 100 ng/mL). We sought to determine the appropriate treatment duration for efficient and cost-effective differentiation protocol for foregut definitive endoderm production in a dynamic suspension culture. Afterward, we evaluated the impact of the initial hPSC aggregate sizes (small: 86 ± 18 μm; medium: 142 ± 32 μm; large: 214 ± 34 μm) as critical bioprocess parameter on differentiation efficacy at the beginning of induction. The results indicated that 1-day priming of hPSCs as 3D aggregates (hPSpheres) with 6 μM CHIR followed by treatment with a low concentration of Activin (10 ng/mL) for 2 days resulted in efficient differentiation to definitive endoderm. This finding confirmed by the presence of ≥70% SOX17/FOXA2-double positive cells that highly expressed the anterior endodermal marker HEX. These endodermal cells differentiated efficiently into mature functional hepatocytes [60% albumin (ALB)-positive cells]. The results showed that the initial size of hPSC aggregates significantly impacted on the efficacy of differentiation. The medium sized-hPSpheres resulted in higher productivity and differentiation efficiency for scalable hepatocytes production, whereas small aggregates resulted in significant cell-loss after CHIR treatment and large aggregates had less efficacious endodermal differentiation. Differentiated cells exhibited multiple characteristics of primary hepatocytes as evidenced by expressions of liver-specific markers, indocyanine green and low-density lipoprotein uptake, and glycogen storage. Thus, this platform could be employed for scalable production of hPSC-derived hepatocytes for clinical and drug discovery applications.

Hepatocellular carcinoma (HCC) is the second leading cause of death due to cancer. Although there are different treatment options, these strategies are not efficient in terms of restricting the tumor cell’s proliferation and metastasis. The liver tumor microenvironment contains the non-parenchymal cells with supportive or inhibitory effects on the cancerous phenotype of HCC. Several signaling pathways are dis-regulated in HCC and cause uncontrolled cell propagation, metastasis, and recurrence of liver carcinoma cells. Recent studies have established new approaches for the prevention and treatment of HCC using small molecules. Small molecules are compounds with a low molecular weight that usually inhibit the specific targets in signal transduction pathways. These components can induce cell cycle arrest, apoptosis, block metastasis, and tumor growth. Devising strategies for simultaneously targeting HCC and the non-parenchymal population of the tumor could lead to more relevant research outcomes. These strategies may open new avenues for the treatment of HCC with minimal cytotoxic effects on healthy cells. This study provides the latest findings on critical signaling pathways governing HCC behavior and using small molecules in the control of HCC both in vitro and in vivo models.

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Derivation of definitive endoderm (DE) from human embryonic stem cells (hESCs) can address the needs of regenerative medicine for endoderm-derived organs such as the pancreas and liver. Fibrous substrates which topographically recapitulate native extracellular matrix have been known to promote the stem cell differentiation. However, the optimal fiber diameter remains to be determined for the desired differentiation. Here, we have developed a simple method to precisely fabricate electrospun poly(ε-caprolactone) fibers with four distinct average diameters at nano- and microscale levels (200, 500, 800, and 1300 nm). Human ESCs were cultured as clumps or single cells and induced into DE differentiation to determine the optimal topography leading to the promoted differentiation compared with planar culture plates. Gene expression analysis of the DE-induced cells showed significant upregulation of DE-specific genes exclusively on the 200-nm fibers. By Western blot analysis, significant expression of DE-specific proteins was found when hESCs were cultured on the 200 nm substrate as single cells rather than clumps, probably due to more efficient cell-matrix interaction realized by morphological observations of the cell colonies. The results indicated that nanofibrillar substrates, only at ultrathin fiber diameters, provided a better environment for DE differentiation of hESC, which holds great promise in prospective tissue engineering applications.