Human amniotic epithelial cells (hAECs) represent an interesting clinical alternative to human embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells in regenerative medicine. The potential of hAECs can be enhanced ex vivo by their partial pre-differentiation. The aim of this study was to evaluate the effectiveness of 18-day differentiation of hAECs into endodermal cells, hepatic precursor cells, and cells showing functional features of hepatocytes using culture media supplemented with high (100 ng/mL) concentrations of EGF or HGF. The cells obtained after differentiation showed changes in morphology and increased expression of AFP, ALB, CYP3A4, CYP3A7, and GSTP1 genes. HGF was more effective than EGF in increasing the expression of liver-specific genes in hAECs. However, EGF stimulated the differentiation process more efficiently and yielded more hepatocyte-like cells capable of synthesizing α-fetoprotein during differentiation. Additionally, after 18 days, GST transferases, albumin, and CYP P450s, which proved their partial functionality, were expressed. In summary, HGF and EGF at a dose of 100 ng/mL can be successfully used to obtain hepatocyte-like cells between days 7 and 18 of hAEC differentiation. However, the effectiveness of this process is lower compared with hiPSC differentiation; therefore, optimization of the composition of the medium requires further research.

Human pluripotent stem cells (hPSCs) hold great promise for future applications in drug discovery and cell therapies. hPSC culture protocols require specific substrates and medium supplements to support cell expansion and lineage specific differentiation. The animal origin of these substrates is a severe limitation when considering the translation of hPSC derivatives to the clinic and in vitro disease modeling. The present study evaluates the use of a human placenta-derived extracellular matrix (ECM) hydrogel, HuGentra^Ⓡ, to support tri-lineage differentiation of human induced pluripotent stem cells (hiPSCs). Lineage-specific embryoid bodies (EBs) were plated onto three separate matrices, and differentiation efficiency was evaluated based on morphology, protein, and gene expression. HuGentra was found to support the differentiation of hiPSCs to all three germ layers: ectodermal, mesodermal, and endodermal lineages. hiPSCs differentiated into neurons, cardiomyocytes, and hepatocytes on HuGentra had similar morphology, protein, and gene expression compared to differentiation on Matrigel or other cell preferred matrices. HuGentra can be considered as a suitable human substrate for hiPSC differentiation.

• Differentiation
• Human ECM
• Tri-lineage
• hiPSC

• liver
• regenerative medicine
• tissue engineering
• translational medicine

• Animals
• Humans
• Liver Diseases/physiopathology
• Liver Diseases/therapy
• Liver Regeneration/physiology
• Regenerative Medicine
• Tissue Engineering
• Tissue Scaffolds/chemistry
• Translational Research, Biomedical

Clinically relevant human induced pluripotent stem cell (hiPSC) derivatives require efficient protocols to differentiate hiPSCs into specific lineages. Here we developed a fully defined xeno-free strategy to direct hiPSCs toward osteoblasts within 21 days. The strategy successfully achieved the osteogenic induction of four independently derived hiPSC lines by a sequential use of combinations of small-molecule inducers. The induction first generated mesodermal cells, which subsequently recapitulated the developmental expression pattern of major osteoblast genes and proteins. Importantly, Col2.3-Cherry hiPSCs subjected to this strategy strongly expressed the cherry fluorescence that has been observed in bone-forming osteoblasts in vivo. Moreover, the protocol combined with a three-dimensional (3D) scaffold was suitable for the generation of a xeno-free 3D osteogenic system. Thus, our strategy offers a platform with significant advantages for bone biology studies and it will also contribute to clinical applications of hiPSCs to skeletal regenerative medicine.

• Hedgehog
• Helioxanthin
• Mesoderm
• Three-dimensional culture
• Wnt

Recent advances in cell therapy have encouraged researchers to provide an alternative for treatment and restoration of damaged liver through using hepatocytes. However, these cells quickly lose their functional capabilities in vitro. Here, we aim to use the secretome of mesenchymal stromal cells (MSCs) to improve in vitro maintenance conditions for hepatocytes.

In this experimental study, following serum deprivation, human adipose tissue-derived MSCs (hAT-MSCs) were cultured for 24 hours under normoxic (N) and hypoxic (H) conditions. Their conditioned media (CM) were subsequently collected and labeled as N-CM (normoxia) and H-CM (hypoxia). Murine hepatocytes were isolated by perfusion of mouse liver with collagenase, and were cultured in hepatocyte basal (William’s) medium supplemented with 4% N-CM or H-CM. Untreated William’s and hepatocyte-specific media (HepZYM) were used as controls. Finally, we evaluated the survival and proliferation rates, as well as functionality and hepatocyte-specific gene expressions of the cells.

We observed a significant increase in viability of hepatocytes in the presence of N-CM and H-CM compared to HepZYM on day 5, as indicated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium) assay. Indocyanine green (ICG) uptake of hepatocytes in the H-CM and HepZYM groups on days 3 and 5 also suggested that H-CM maintained the hepatocytes at about the same level as the hepatocyte-specific medium. The HepZYM group had significantly higher levels of albumin (Alb) and urea secretion compared to the other groups (P<0.0001). However, there were no significant differences in cytochrome activity and cytochrome gene expression profiles among these groups. Finally, we found a slightly, but not significantly higher concentration of vascular endothelial growth factor (VEGF) in the H-CM group compared to the N-CM group (P=0.063).

The enrichment of William’s basal medium with 4% hAT-MSC-H-CM improved some physiologic parameters in a primary hepatocyte culture.

• Conditioned Medium
• Mesenchymal Stromal Cell
• Primary Hepatocyte
• Regenerative Medicine

• aggregate
• definitive endoderm
• hepatocyte
• scale-up differentiation
• size-controlled differentiation

Hepatocyte-like cells (HLCs) are generated from either various human pluripotent stem cells (hPSCs) including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), or direct cell conversion, mesenchymal stem cells as well as other stem cells like gestational tissues. They provide potential cell sources for biomedical applications. Liver transplantation is the gold standard treatment for the patients with end stage liver disease, but there are many obstacles limiting this process, like insufficient number of donated healthy livers. Meanwhile, the number of patients receiving a liver organ transplant for a better life is increasing. In this regard, HLCs may provide an adequate cell source to overcome these shortages. New molecular engineering approaches such as CRISPR/ Cas system applying in iPSCs technology provide the basic principles of gene correction for monogenic inherited metabolic liver diseases, as another application of HLCs. It has been shown that HLCs could replace primary human hepatocytes in drug discovery and hepatotoxicity tests. However, generation of fully functional HLCs is still a big challenge; several research groups have been trying to improve current differentiation protocols to achieve better HLCs according to morphology and function of cells. Large-scale generation of functional HLCs in bioreactors could make a new opportunity in producing enough hepatocytes for treating end-stage liver patients as well as other biomedical applications such as drug studies. In this review, regarding the biomedical value of HLCs, we focus on the current and efficient approaches for generating hepatocyte-like cells in vitro and discuss about their applications in regenerative medicine and drug discovery.

• Cell Therapy
• Drug Discovery
• Gene Therapy
• Hepatocyte

Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models.

• cell therapy
• cell transplantation
• disease-specific induced pluripotent stem cells
• human liver disease modeling
• liver disease
• metabolic liver disease
• regenerative medicine

Embryonic stem cells (ESCs) are pluripotent cells with great therapeutic potentials. The in vitro differentiation of ESC was designed by recapitulating embryogenesis. Significant progress has been made to improve the in vitro differentiation protocols by toning soluble maintenance factors. However, more robust methods for lineage-specific differentiation and maturation are still under development. Considering the complexity of in vivo embryogenesis environment, extracellular matrix (ECM) cues should be considered besides growth factor cues. ECM proteins bind to cells and act as ligands of integrin receptors on cell surfaces. Here, we summarize the role of the ECM and integrins in the formation of three germ layer progenies. Various ECM–integrin interactions were found, facilitating differentiation toward definitive endoderm, hepatocyte-like cells, pancreatic beta cells, early mesodermal progenitors, cardiomyocytes, neuroectoderm lineages, and epidermal cells, such as keratinocytes and melanocytes. In the future, ECM combinations for the optimal ESC differentiation environment will require substantial study.

• differentiation
• embryonic stem cells
• extracellular matrix
• integrin
• microenvironment
• regeneration

Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors (GFs), cytokines, transcription factors (TFs), hormones, oxidative stress products, metabolic networks, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentiation, causing them to lose hepatocyte function. For this reason, most studies focus on inducing stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), hepatic progenitor cells (HPCs), and mesenchymal stem cells (MSCs), to differentiate into hepatocyte-like cells (HLCs) in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to maintain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regeneration. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

AIM: To present a novel 3-step procedure to efficiently direct the differentiation of mouse embryonic stem cells (ESCs) into hepatocytes.

METHODS: Mouse ESCs were first induced to differentiate into definitive endoderm cells by three days of activin A treatment. Next, definitive endoderm cells were induced to efficiently differentiate to hepatocytes in the presence of acid fibroblast growth factor (aFGF) and trichostatin A (TSA) in the culture medium for 5 d.

RESULTS: After 10 d of further in vitro maturation, the morphological and phenotypic markers of hepatocytes were characterized using light microscopy, immunofluorescence and RT-PCR. Furthermore, these cells were tested for the functions associated with mature hepatocytes including glycogen storage, indocyanine green uptake and release, and the rate of hepatic differentiation was determined by counting the albumin-positive cells, which showed that the rate of hepatic differentiation was 57.38%.

CONCLUSION: The method presented in this study provides a new resource for hepatocyte transplantation.

• Embryonic stem cells
• Hepatocyte
• Induced differentiation

Liver cancer is an aggressive disease with a high mortality rate. Management of liver cancer is strongly dependent on the tumor stage and underlying liver disease. Unfortunately, most cases are discovered when the cancer is already advanced, missing the opportunity for surgical resection. Thus, an improved understanding of the mechanisms responsible for liver cancer initiation and progression will facilitate the detection of more reliable tumor markers and the development of new small molecules for targeted therapy of liver cancer. Recently, there is increasing evidence for the “cancer stem cell hypothesis”, which postulates that liver cancer originates from the malignant transformation of liver stem/progenitor cells (liver cancer stem cells). This cancer stem cell model has important significance for understanding the basic biology of liver cancer and has profound importance for the development of new strategies for cancer prevention and treatment. In this review, we highlight recent advances in the role of liver stem cells in hepatocarcinogenesis. Our review of the literature shows that identification of the cellular origin and the signaling pathways involved is challenging issues in liver cancer with pivotal implications in therapeutic perspectives. Although the dedifferentiation of mature hepatocytes/cholangiocytes in hepatocarcinogenesis cannot be excluded, neoplastic transformation of a stem cell subpopulation more easily explains hepatocarcinogenesis. Elimination of liver cancer stem cells in liver cancer could result in the degeneration of downstream cells, which makes them potential targets for liver cancer therapies. Therefore, liver stem cells could represent a new target for therapeutic approaches to liver cancer in the near future.

• Liver cancer
• Liver stem cells
• Hepatocarcinogenesis
• Tumorigenic transformation
• Transdifferentiation