Chromatin remodeling is restricted by transient GATA6 binding during iPSC differentiation to definitive endoderm.

In addition to cooperatively driving transcriptional programs, emerging evidence supports transcription factors interacting with one another to modulate the outcome of binding events. As such, transcription factor interactions fine-tune the unique gene expression profiles required for developmental progression. Using human-induced pluripotent stem cells as a model of human endoderm lineage commitment, we reveal that GATA6 transiently co-localizes with EOMES at regions associated with non-endodermal lineages and is required for the repression of chromatin opening at these loci. Our results indicate that GATA6-dependent repression of chromatin remodeling, which is potentially mediated via the recruitment of NCOR1 to the EOMES interactome, contributes to definitive endoderm commitment. We anticipate that similar mechanisms are common during human development, furthering our understanding of the complex mechanisms that define cell fate decisions.

GATA6 defines endoderm fate by controlling chromatin accessibility during differentiation of human-induced pluripotent stem cells.

In addition to driving specific gene expression profiles, transcriptional regulators are becoming increasingly recognized for their capacity to modulate chromatin structure. GATA6 is essential for the formation of definitive endoderm; however, the molecular basis defining the importance of GATA6 to endoderm commitment is poorly understood. The members of the GATA family of transcription factors have the capacity to bind and alter the accessibility of chromatin. Using pluripotent stem cells as a model of human development, we reveal that GATA6 is integral to the establishment of the endoderm enhancer network via the induction of chromatin accessibility and histone modifications. We additionally identify the chromatin-modifying complexes that interact with GATA6, defining the putative mechanisms by which GATA6 modulates chromatin architecture. The identified GATA6-dependent processes further our knowledge of the molecular mechanisms that underpin cell-fate decisions during formative development.

Generation of isogenic Propionyl-CoA carboxylase beta subunit (PCCB) deficient induced pluripotent stem cell lines.

Propionic acidemia (PA) is an autosomal recessive metabolic liver disease caused by defects in propionyl CoA carboxylase. Propionyl CoA carboxylase is a dodecameric enzyme consisting of multiple copies of alpha and beta subunits encoded by the PCCA and PCCB genes. Mutations in either PCCA or PCCB can cause the disease. PA is categorized as a rare disease and accessing patients' cells to study the disease has been challenging. Here we describe the generation of two isogenic induced pluripotent stem cell (iPSC) lines in which exon 2 of the PCCB gene was mutated using CRISPR Cas9 gene editing. The PCCB-/- iPSCs express characteristic pluripotency proteins, are competent to differentiate into cell lineages from each of the three embryonic germ layers and display a normal karyotype.

A simple and cost-efficient adherent culture platform for human gastric primary cells, as an in vitro model for Helicobacter pylori infection.

BACKGROUND: Most two- dimensional in vitro models for studying host- H. pylori interactions rely on tumor-derived cell lines, which harbor malignant alterations. The recent development of human gastric organoids has overcome this limitation and provides a highly sophisticated, yet costly, short-term model for H. pylori infection, with restricted use in low-budget centers. METHOD: Tissue specimens from upper, middle, and lower stomachs of H. pylori-negative volunteers were collectively dispersed and cultured on mouse embryonic fibroblast (MEF) or collagen-coated plates. Gastric primary cells (GPCs) were evaluated by light microscopy, immunostaining, qRT-PCR and ELISA analysis of cellular secretions, before and after H. pylori infection. RESULTS: The formation and long-term (up to 1 year) maintenance of GPCs was highly dependent on adherent inactivated MEF cells, cultured in enriched media. These cells were multipassageable and able to undergo stable freezer storage and subsequent revival. The cellular composition of GPCs included the combination of cytokeratin 18 (CK18) and E-cadherin (E-cad)-positive epithelial cells, MUC5AC-positive gastric cells, and leucine-rich repeat containing G protein-coupled receptor 5 (LGR5)-positive progenitor cells. These cells produced significant amounts of gastric pepsinogens I and II. GPCs also allowed for extended (up to 96 hours) H. pylori infection, during which they underwent morphological alterations (cellular vacuolation and elongation) and hyperproduction of gastric pepsinogens and inflammatory cytokines (IL-8 and TNF-α). CONCLUSION: We, hereby, present a simple, consistent, and cost-efficient gastric cell culture system, which provides a suitable model for extended in vitro infection of H. pylori. This platform can be employed for a variety of gastric-related research.

Three-dimensional liver-derived extracellular matrix hydrogel promotes liver organoids function.

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.

Modeling Inborn Errors of Hepatic Metabolism Using Induced Pluripotent Stem Cells.

Inborn errors of hepatic metabolism are because of deficiencies commonly within a single enzyme as a consequence of heritable mutations in the genome. Individually such diseases are rare, but collectively they are common. Advances in genome-wide association studies and DNA sequencing have helped researchers identify the underlying genetic basis of such diseases. Unfortunately, cellular and animal models that accurately recapitulate these inborn errors of hepatic metabolism in the laboratory have been lacking. Recently, investigators have exploited molecular techniques to generate induced pluripotent stem cells from patients' somatic cells. Induced pluripotent stem cells can differentiate into a wide variety of cell types, including hepatocytes, thereby offering an innovative approach to unravel the mechanisms underlying inborn errors of hepatic metabolism. Moreover, such cell models could potentially provide a platform for the discovery of therapeutics. In this mini-review, we present a brief overview of the state-of-the-art in using pluripotent stem cells for such studies.

In Vitro Generated Hepatocyte-Like Cells: A Novel Tool in Regenerative Medicine and Drug Discovery.

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.

Effect of Secreted Molecules of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells on Acute Hepatic Failure Model.

Adult tissue-derived mesenchymal stem cells (MSCs) show tremendous promise for a wide array of therapeutic applications predominantly through paracrine activity. Recent reports showed that human embryonic stem cell (ESC)-derived MSCs are an alternative for regenerative cellular therapy due to manufacturing large quantities of MSCs from a single donor. However, no study has been reported to uncover the secretome of human ESC-MSCs as treatment of an acute liver failure (ALF) mouse model. We demonstrated that human ESC-MSCs showed similar morphology and cell surface markers compared with bone marrow-derived MSCs. ESC-MSCs exhibited a higher growth rate during early in vitro expansion, along with adipogenic and osteogenic differentiation potential. Treatment with ESC-MSC-conditioned medium (CM) led to statistically significant enhancement of primary hepatocyte viability and increased immunomodulatory interleukin-10 secretion from lipopolysaccharide-induced human blood mononuclear cells. Analysis of the MSCs secretome by a protein array screen showed an association between higher frequencies of secretory proteins such as vascular endothelial growth factor (VEGF) and regulation of cell proliferation, cell migration, the development process, immune system process, and apoptosis. In this thioacetamide-induced mouse model of acute liver injury, we observed that systemic infusion of VEGF led to significant survival. These data have provided the first experimental evidence of the therapeutic potential of human ESC-MSC-derived molecules. These molecules show trophic support to hepatocytes, which potentially creates new avenues for the treatment of ALF, as an inflammatory condition.

Forced expression of Hnf4a induces hepatic gene activation through directed differentiation.

Embryonic stem (ES) cells are capable of unlimited self-renewal and have a diverse differentiation potential. These unique features make ES cells as an attractive source for developmental biology studies. Having the mature hepatocyte in the lab with functional activities is valuable in drug discovery studies. Overexpression of hepatocyte lineage-specific transcription factors (TFs) becomes a promising approach in pluripotent cell differentiation toward liver cells. Many studies generate transgenic ES cell lines to examine the effects of specific TFs overexpression in cell differentiation. In the present report, we have addressed whether a suspension or adherent model of differentiation is an appropriate way to study the role of Hnf4a overexpression. We generated ES cells that carried a doxycycline (Dox)-inducible Hnf4a using lentiviral vectors. The transduced cells were subjected to induced Hnf4a overexpression through both spontaneous and directed differentiation methods. Gene expression analysis showed substantially increased expression of hepatic gene markers, particularly Ttr and endogenous Hnf4a, in transduced cells differentiated by the directed approach. These results demonstrated that forced expression of TFs during directed differentiation would be an appropriate way to study relevant gene activation and the effects of overexpression in the context of hepatic differentiation.

Forced expression of Hnf1b/Foxa3 promotes hepatic fate of embryonic stem cells.

Embryonic stem (ES) cell-derived hepatocytes have the potential to be used for basic research, regenerative medicine, and drug discovery. Recent reports demonstrated that in addition to conventional differentiation inducers such as chemical compounds and cytokines, overexpression of lineage-specific transcription factors could induce ES cells to differentiate to a hepatic fate. Here, we hypothesized that lentivirus-mediated inducible expression of hepatic lineage transcription factors could enhance mouse ES cells to hepatocyte-like cells. We screened the effects of candidate transcription factors Hnf1b, Hnf1a, Hnf4a, Foxa1, Foxa3 and Hex, and determined that the combination of Hnf1b/Foxa3 promoted expression of several hepatic lineage-specific markers and proteins, in addition to glycogen storage, ICG uptake, and secretion of albumin and urea. The differentiated cells were engraftable and expressed albumin when transplanted into a carbon tetrachloride-injured mouse model. These results demonstrated the crucial role of Hnf1b and Foxa3 in hepatogenesis in vitro and provided a valuable tool for the efficient differentiation of HLCs from ES cells.

Enhanced direct conversion of fibroblasts into hepatocyte-like cells by Kdm2b.

Cell fate conversion of terminally differentiated cells by defined transcription factors between different lineages is a new approach to produce new cells that have the capability to repair or replace diseased and damaged tissues. Previous studies have demonstrated that this inefficient process can be facilitated by the inclusion of additional factors. Here we report that Kdm2b, a histone demethylase, has the capability to promote conversion of fibroblasts to functional induced hepatocyte-like (iHep) cells in combination with previously reported hepatic lineage transcription factors, Hnf4α and Foxa3. This approach led to increased numbers of epithelial-like colonies, hepatic markers and functionality which included periodic acid-Schiff (PAS) positive colonies, CYP450 activity, low-density lipoprotein and indocyanine green (ICG) uptake, as well as Albumin secretion. Additionally, the transplanted iHep cells were engraftable, expressed Albumin, and contributed to the recovery of a carbon tetrachloride-injured mouse model. These results have not only identified an important epigenetic factor for iHep generation, but also brought new insight into the molecular nature of hepatogenesis and future biomedical applications for liver diseases.

Transcription factor-mediated reprograming of fibroblasts to hepatocyte-like cells.

Direct conversion by overexpression of defined transcription factors (TFs) is a promising new method that can generate desired cell types from abundant, accessible cells. While previous studies have reported hepatic generation from fibroblasts, tremendous interest exists in the understanding of hepatic reprograming and its applicability in regenerative medicine. Here, we show that overexpression of Yamanaka factors can induce reprograming of mouse fibroblasts into cells that closely resemble hepatocytes in vitro in the presence of an optimized hepatic growth medium. By screening the effects of 20 candidate transcription factors, we identified a combination of three TFs (Hnf4a, Cebpa, and Nr1i2) that can convert fibroblasts into a hepatic fate. These factors in conjunction with Yamanaka factors increase the efficiency of hepatic reprograming. The induced hepatocyte-like (iHep) cells have multiple hepatocyte-specific characteristics; express hepatocyte-specific markers, glycogen storage, albumin secretion, urea production, as well as low-density lipoprotein and indocyanin green uptake. Production of iHep cells by these novel approaches may bring new insights into the molecular nature of hepatocyte differentiation and future cell-based therapeutics for liver diseases.

Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells.

Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.

Differentiation of human embryonic stem cells to hepatocyte-like cells on a new developed xeno-free extracellular matrix.

Human embryonic stem cells (hESCs) provide a new source for hepatocyte production in translational medicine and cell replacement therapy. The reported hESC-derived hepatocyte-like cells (HLCs) were commonly generated on Matrigel, a mouse cell line-derived extracellular matrix (ECM). Here, we performed the hepatic lineage differentiation of hESCs following a stepwise application of growth factors on a newly developed serum- and xeno-free, simple and cost-benefit ECM, designated "RoGel," which generated from a modified conditioned medium of human fibroblasts. In comparison with Matrigel, the differentiated HLCs on both ECMs expressed similar levels of hepatocyte-specific genes, secreted α-fetoprotein, and metabolized ammonia, showed glycogen storage activity as well as low-density lipoprotein and indocyanine green uptake. The transplantation of hESC-HLCs into the carbon tetrachloride-injured liver demonstrated incorporation of the cells into the host mouse liver and the expression of albumin. The results suggest that the xeno-free and cost-benefit matrix may be applicable in bioartificial livers and also may facilitating a clinical application of human pluripotent stem cell-derived hepatocytes in the future.

Generation of functional hepatocyte-like cells from human pluripotent stem cells in a scalable suspension culture.

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.

Therapeutic potential of human induced pluripotent stem cell-derived mesenchymal stem cells in mice with lethal fulminant hepatic failure.

Large-scale production and noninvasive methods for harvesting mesenchymal stem cells (MSCs), particularly in elderly individuals, has prompted researchers to find new patient-specific sources for MSCs in regenerative medicine. This study aims to produce MSCs from human induced pluripotent stem cells (hiPSCs) and to evaluate their therapeutic effects in a CCl4-induced mouse model of fulminant hepatic failure (FHF). hiPSC-MSCs have shown MSC morphology, antigen profile and differentiation capabilities, and improved hepatic function in our model. hiPSC-MSC-transplanted animals provide significant benefit in terms of survival, serum LDH, total bilirubin, and lipid peroxidation. hiPSC-MSC therapy resulted in a one-third reduction of histologic activity index and a threefold increase in the number of proliferating hepatocytes. This was accompanied by a significant decrease in the expression levels of collagen type I, Mmp13, Mmp2, and Mmp9 genes and increase in Timp1 and Timp2 genes in transplanted groups. hiPSC-MSCs secreted hepatocyte growth factor (HGF) in vitro and also expressed HGF in evaluated liver sections. Similar results were observed with human bone marrow (hBM)-derived MSCs. In conclusion, our results have demonstrated that hiPSC-MSCs might be valuable appropriate alternatives for hBM-MSCs in FHF liver repair and support liver function by cell therapy with a large-scale production capacity, patient-specific nature, and no invasive MSC harvesting.

Repeated versus single transplantation of mesenchymal stem cells in carbon tetrachloride-induced liver injury in mice.

Despite its numerous limitations, liver transplants are the only definite cure for end-stage liver disease. Various stem cell populations may contribute to liver regeneration, of which there is accumulating evidence of the contribution of mesenchymal stem cells (MSCs). This study examines the hypothesis that repeated infusions of human bone marrow-derived MSCs (hBMMSCs)can improve liver injury in an experimental model. MSCs were intravenously transplanted into immunosuppressed mice with carbon tetrachloride (CCl(4))-induced liver fibrosis. Transplanting 3x10(6) MSCs in three divided doses improved survival,liver fibrosis and necrosis compared with injection of the same number of MSCs in a single dose. This was accompanied by increased influence on the expression of the fibrogenic/fibrolytic related genes Col1a1, Timp1 and Mmp13 in the repeated transplant group. Repeat administration of MSCs was three times more effective in homing of PKH-tagged transplanted cells 3 weeks post-transplant compared with the single transplant group. The benefits of repeated transplants may be of considerable significance in clinical trials on liver failure.

Differentiation and transplantation of human induced pluripotent stem cell-derived hepatocyte-like cells.

The generation of human induced pluripotent stem cells (hiPSCs) with a high differentiation potential provided a new source for hepatocyte generation not only for drug discovery and in vitro disease models, but also for cell replacement therapy. However, the reported hiPSC-derived hepatocyte-like cells (HLCs) were not well characterized and their transplantation, as the most promising clue of cell function was not reported. Here, we performed a growth factor-mediated differentiation of functional HLCs from hiPSCs and evaluated their potential for recovery of a carbon tetrachloride (CCl4)-injured mouse liver following transplantation. The hiPSC-derived hepatic lineage cells expressed hepatocyte-specific markers, showed glycogen and lipid storage activity, secretion of albumin (ALB), alpha-fetoprotein (AFP), urea, and CYP450 metabolic activity in addition to low-density lipoprotein (LDL) and indocyanin green (ICG) uptake. Similar results were observed with human embryonic stem cell (hESC)-derived HLCs. The transplantation of hiPSC-HLCs into a CCl4-injured liver showed incorporation of the hiPSC-HLCs into the mouse liver which resulted in a significant enhancement in total serum ALB after 1 week. A reduction of total serum LDH and bilirubin was seen when compared with the control and sham groups 1 and 5 weeks post-transplantation. Additionally, we detected human serum ALB and ALB-positive transplanted cells in both the host serum and livers, respectively, which showed functional integration of transplanted cells within the mouse livers. Therefore, our results have opened up a proof of concept that functional HLCs can be generated from hiPSCs, thus improving the general condition of a CCl4-injured mouse liver after their transplantation. These results may bring new insights in the clinical applications of hiPSCs once safety issues are overcome.