Generation Of Hepatocyte-like Cells Research Articles

A robust reprogramming strategy for generating hepatocyte-like cells usable in pharmaco-toxicological studies

BackgroundHigh-throughput pharmaco-toxicological testing frequently relies on the use of established liver-derived cell lines, such as HepG2 cells. However, these cells often display limited hepatic phenotype and features of neoplastic transformation that may bias the interpretation of the results. Alternate models based on primary cultures or differentiated pluripotent stem cells are costly to handle and difficult to implement in high-throughput screening platforms. Thus, cells without malignant traits, optimal differentiation pattern, producible in large and homogeneous amounts and with patient-specific phenotypes would be desirable.MethodsWe have designed and implemented a novel and robust approach to obtain hepatocytes from individuals by direct reprogramming, which is based on a combination of a single doxycycline-inducible polycistronic vector system expressing HNF4A, HNF1A and FOXA3, introduced in human fibroblasts previously transduced with human telomerase reverse transcriptase (hTERT). These cells can be maintained in fibroblast culture media, under standard cell culture conditions.ResultsClonal hTERT-transduced human fibroblast cell lines can be expanded at least to 110 population doublings without signs of transformation or senescence. They can be easily differentiated at any cell passage number to hepatocyte-like cells with the simple addition of doxycycline to culture media. Acquisition of a hepatocyte phenotype is achieved in just 10 days and requires a simple and non-expensive cell culture media and standard 2D culture conditions. Hepatocytes reprogrammed from low and high passage hTERT-transduced fibroblasts display very similar transcriptomic profiles, biotransformation activities and show analogous pattern behavior in toxicometabolomic studies. Results indicate that this cell model outperforms HepG2 in toxicological screening. The procedure also allows generation of hepatocyte-like cells from patients with given pathological phenotypes. In fact, we succeeded in generating hepatocyte-like cells from a patient with alpha-1 antitrypsin deficiency, which recapitulated accumulation of intracellular alpha-1 antitrypsin polymers and deregulation of unfolded protein response and inflammatory networks.ConclusionOur strategy allows the generation of an unlimited source of clonal, homogeneous, non-transformed induced hepatocyte-like cells, capable of performing typical hepatic functions and suitable for pharmaco-toxicological high-throughput testing. Moreover, as far as hepatocyte-like cells derived from fibroblasts isolated from patients suffering hepatic dysfunctions, retain the disease traits, as demonstrated for alpha-1-antitrypsin deficiency, this strategy can be applied to the study of other cases of anomalous hepatocyte functionality.

Raman Spectroscopic Characterization of Hepatic Differentiation of Mesenchymal Stem Cells

Background and Aims: Mesenchymal stem cells (MSCs) are a preferred cell source for the generation of hepatocyte-like cells in regenerative medicine. They can be isolated from different sources, including adipose tissues. The Raman spectroscopy approach was evaluated for quick and efficient identification of MSCs differentiation status and a broader perspective on cell differentiation. Materials and Methods: The human adipose-derived mesenchymal stem cells (hASCs) were differentiated toward hepatocyte-like cells using a well-established method. The cells were cultured on fluorescence-free quartz discs, and the efficiency of differentiation was examined using molecular and biochemical methods. The Raman spectra were recorded at days 1, 7, 14, and 21 of differentiation, and HepG2 was used as a positive control. Results: The changes in Raman spectra were detected during the sequential stages of differentiation, and the pattern of peaks on the last day of differentiation was remarkably similar to the positive control (HepG2). Conclusion: Raman spectroscopy showed considerable potential to characterize hepatic differentiation.

Regenerative medicine for the hepatobiliary system: A review.

Liver transplantation, the only proven treatment for end-stage liver disease and acute liver failure, is hampered by the scarcity of donors. Regenerative medicine provides an alternative therapeutic approach. Tremendous efforts dedicated to liver regenerative medicine include the delivery of transplantable cells, microtissues, and bioengineered whole livers via tissue engineering and the maintenance of partial liver function via extracorporeal support. This brief review summarizes the current status of regenerative medicine for the hepatobiliary system. For liver regenerative medicine, the focus is on strategies for expansion of transplantable hepatocytes, generation of hepatocyte-like cells, and therapeutic potential of engineered tissues in liver disease models. For biliary regenerative medicine, the discussion concentrates on the methods for generation of cholangiocyte-like cells and strategies in the treatment of biliary disease. Significant advances have been made in large-scale and long-term expansion of liver cells. The development of tissue engineering and stem cell induction technology holds great promise for the future treatment of hepatobiliary diseases. The application of regenerative medicine in liver still lacks extensive animal experiments. Therefore, a large number of preclinical studies are necessary to provide sufficient evidence for their therapeutic effectiveness. Much remains to be done for the treatment of hepatobiliary diseases with regenerative medicine.

Generation of hepatocyte-like cells from human urinary epithelial cells and the role of autophagy during direct reprogramming

Somatic cells can be directly reprogrammed into other cell lineages, which holds great promise for regenerative medicine. However, low efficiency and obscure mechanism hinder the application of direct reprogramming. Here, we show that overexpressing the hepatic-specific transcription factors (TFs) HNF1α, FOXA3, and GATA4 was sufficient to convert human urinary epithelial cells (hUCs) into induced hepatocyte-like cells (iHeps). The obtained iHeps were confirmed to express various hepatocyte-specific genes with multiple mature hepatocyte functions. Moreover, autophagy-related genes P62, ULK1, BECN1, VPS34, and LC3B were upregulated in the early stage of reprogramming and knockout of P62 and BECN1 in hUCs with CRISPR/Cas9 technology increased the efficiency of direct reprogramming. Collectively, we established a non-invasive approach to convert hUCs into iHeps and provided a glimpse into the role of autophagy in this process.

Advances in Human Induced Pluripotent Stem Cell-Derived Hepatocytes for Use in Toxicity Testing

Induced pluripotent stem cells (iPSCs) can be differentiated into multiple cell types in the body while maintaining proliferative capabilities. The generation of hepatocyte-like cells (HLCs) from iPSCs has resulted in a new source for liver cells. Since healthy primary human hepatocytes and hepatic cells are difficult to obtain, HLCs are gaining attention. HLCs can be obtained from a continuous, stable source while maintaining their original donor genotype, which opens new avenues into patient-specific testing and therapeutics. Studies have utilized HLCs for toxicity testing to further understand their drug metabolizing capabilities. This review focuses on advances being made to achieve hepatic functions from HLCs, their current use in hepatotoxicity testing, and their potential for future liver-related toxicity evaluations.

Generation of Hepatocyte-Like Cells by Different Strategies for Liver Regeneration

Incidence and mortality of liver disease has increased globally in recent years. Orthotopic liver transplantation is a well-developed, effective therapy even for the end-stage liver diseases. However, the application of the technique is limited by the short supply of donors and the complexity of orthotopic liver transplantation. Therefore, hepatocyte transplantation as a new therapeutic option was developed. Unfortunately, the routine supply of high quality human hepatocytes was also restricted and the liver donor was lacking, too. Thus, exploration and study for available and renewable sources of nondonor hepatocytes are both necessary and important. Researches have proved that nondonor hepatocyte-like cells can be derived from various types of cells including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), liver stem cells (LSCs), fibroblasts by lineage reprogramming, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). In this review, we describe recent advances in the field of the cell therapy in liver regeneration. In addition, we also discuss the existing restrictions in these types of therapies and ongoing developments.

Cell fate modification toward the hepatic lineage by extrinsic factors.

The lineage of a somatic cell can be altered by targeting its signaling networks with small molecules and/or genetically altering the expression of key transcription factors. Depending on the combination of factors, fibroblasts can be fully reprogrammed into induced pluripotent stem (iPS) cells or directly converted into specific cell lineages, bypassing the pluripotent state. The generation of defined target cells will enormously benefit patients who require cell transplantation therapy. In the decade, since iPS cells were first generated, many cell types have been induced from fibroblasts by direct conversion, including hepatocytes. Converted hepatocyte-like cells have been shown to repopulate liver tissues after transplantation in mouse liver disease models, suggesting promise for future application in humans. Thus, to realize safe and efficient cell transplantation therapy, various methods for generating hepatocyte-like cells are being developed. In this review, we summarize the current methods for the generation of hepatocyte-like cells via cell fate modification using extrinsic factors.

Generation of functional hepatocyte-like cells from human deciduous periodontal ligament stem cells.

Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases.

Concise Review: Advances in Generating Hepatocytes from Pluripotent Stem Cells for Translational Medicine.

The liver is one of the major organs in the human body. Severe or prolonged exposure of the liver to different factors may cause life‐threatening disease, which necessitates donor organ transplantation. While orthotopic liver transplantation can be used to effectively treat liver failure, it is an invasive procedure, which is severely limited by organ donation. Therefore, alternative sources of liver support have been proposed and studied. This includes the use of pluripotent stem cell‐derived hepatocytes as a renewable source of cells for therapy. In addition to cell‐based therapies, in vitro engineered liver tissue provides powerful models for human drug discovery and disease modeling. This review focuses on the generation of hepatocyte‐like cells from pluripotent stem cells and their application in translational medicine. Stem Cells 2016;34:1421–1426

Efficient generation of hepatocyte-like cells from rat bone marrow mesenchymal stem cells in vitro

Recent studies indicated that stem-cell–derived hepatocyte-like cells are superior to stem cells in therapy for liver failure. Our aim is to present a modified one-step protocol for high-efficiency in vitro generation of hepatocyte-like cells from rat bone marrow mesenchymal stem cells (rBMSCs). rBMSCs were cultured under optimal differentiation conditions. Hepatic differentiation was evaluated by light microscopy (for morphological analysis), reverse-transcription polymerase chain reaction (for expression of hepatocyte-specific genes), and immunocytochemical and immunofluorescence analyses of hepatic proteins, such as alpha-fetoprotein and albumin. Functional assays comprised periodic acid-Schiff staining, supernatant urea assay, and albumin radioimmunoassay. The results show that differentiated cells exhibited characteristic hepatocyte morphology, expressed hepatocyte-related genes (as shown by reverse-transcription polymerase chain reaction), and displayed antibody-detectable expressions of alpha-fetoprotein (100% at day 8) and albumin (>70% at 2 weeks). Most differentiated hepatocyte-like cells showed evidence of glycogen synthesis and storage, as shown by periodic acid-Schiff staining. Albumin and urea were detected in supernatants. Our one-step protocol induced the efficient differentiation of rBMSCs into functional hepatocyte-like cells in a two-dimensional in vitro model and may be useful for cell transplantation therapy for liver failure.

643 Mutations in Cel and Hras1 Are Associated With Obesity-Associated Hepatocellular Carcinoma

Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (hiPSCs), with their potential capacity to phenotypically model the influence of genetic variation, could be a useful in vitro hepaticmodel to aidmechanistic understanding of drug-induced liver injury in humans. However, the generation of HLCs with a functionally similar phenotype to the gold standard in vitro model of human primary hepatocytes remain elusive. Furthermore there is a significant variation reported for the differentiation propensities of various hiPSC lines towards the hepatic lineage, although the precise reasons are unclear. Recent reports have highlighted the potential importance of the parental cell used for cellular reprogramming, as hiPSCs may retain an epigenetic memory of their original cell type, with a propensity to differentiate back to its lineage of origin. Therefore, we hypothesize that HLCs generated from hepatocyte-derived hiPSCs may exhibit an improved functional phenotype compared to non-hepatocyte-derived hiPSCs. To control for genotypic differences, variations in the reprogramming techniques, and differences in the hepatic differentiation protocols, we derived hiPSC lines from primary hepatocytes and dermal fibroblasts from the same donors using a non-integrative reprogrammingmethod, and generated HLCs using the same differentiation protocol. The hepatocyte-derived hiPSC (H-hiPSC) lines and dermal fibroblast-derived hiPSC (F-hiPSC) lines successfully generated from the same donors were positive for the pluripotency markers OCT4, SOX2, NANOG, TRA-1-60 and SSEA4, and could spontaneously differentiate into cells of all three germ layers. HLCs were differentiated from all the hiPSC lines and were shown to be positive for the endodermal lineage-specific markers of SOX17, HNF4A and ALB. The HLCs were generated using hiPSCs at low passage numbers (<10) to avoid any loss of epigenetic memory which could arise as a result of continued passage in culture. Separate cultures of HLCs generated from the H-hiPSC and F-hiPSC lines were compared against the donors' primary hepatocytes using real-time PCR for expression of liver-related genes, immunoblotting and assays of key hepatic metabolic function. Our resource of having H-hiPSC and F-hiPSC lines to generate HLCs, with the availability of primary hepatocytes from the same donor as the most appropriate comparator, allows for a definitive assessment of the role of epigenetic memory of the cell origin used for cellular reprogramming, in determining the propensity for hepatic differentiation.

Application of Induced Pluripotent Stem Cells in Liver Diseases.

Tens of millions of patients are affected by liver disease worldwide. Many of these patients can benefit from therapy involving hepatocyte transplantation. Liver transplantation is presently the only proven treatment for many medically refractory liver diseases including end-stage liver failure and inherited metabolic liver disease. However, the shortage in transplantable livers prevents over 40% of listed patients per year from receiving a liver transplant; many of these patients die before receiving an organ offer or become too sick to transplant. Therefore, new therapies are needed to supplement whole-organ liver transplantation and reduce mortality on waiting lists worldwide. Furthermore, the remarkable regenerative capacity of hepatocytes in vivo is exemplified by the increasing number of innovative cell-based therapies and animal models of human liver disorders. Induced pluripotent stem cells (iPSCs) have similar properties to those of embryonic stem cells (ESCs) but bypass the ethical concerns of embryo destruction. Therefore, generation of hepatocyte-like cells (HLCs) using iPSC technology may be beneficial for the treatment of severe liver diseases, screening of drug toxicities, basic research of several hepatocytic disorders, and liver transplantation. Here we briefly summarize the growing number of potential applications of iPSCs for treatment of liver disease.

Generation of hepatocyte-like cells from human induced pluripotent stem (iPS) cells by co-culturing embryoid body cells with liver non-parenchymal cell line TWNT-1.

To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from human iPS cells those show the morphologic and phenotypic properties of primary human hepatocytes. An experimental study. Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011. Human iPS cells were generated and maintained on ES qualified matrigel coated plates supplemented with mTeSR medium or alternatively on mitotically inactivated MEF feeder layer in DMEM/F12 medium containing 20% KOSR, 4ng/ml bFGF-2, 1 x 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mM L-glutamine and 1% penicillin-streptomycin. iPS cells were differentiated to HLCs by sequential culture using a four step differentiation protocol: (I) Generation of embryoid bodies (EBs) in suspension culture; (II) Induction of definitive endoderm (DE) from 2 days old EBs by growth in human activin-A (100 ng/ml) and basic fibroblasts growth factor (bFGF2) (100 ng/ml) on matrigel coated plates; (III) Induction of hepatic progenitors by co-culture with non-parenchymal human hepatic stellate cell line (TWNT-1); and (IV) Maturation by culture in dexamethasone. Characterization was performed by RT-PCR and functional assays. The generated HLCs showed microscopically morphological phenotype of human hepatocytes, expressed liver-specific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia. Functional HLCs were generated from human iPS cells, which could be used for autologus hepatocyte transplantation for liver failure and as in vitro model for determining the metabolic and toxicological properties of drug compounds.

Robust generation of hepatocyte-like cells from human embryonic stem cell populations

Robust generation of hepatocyte-like cells from human embryonic stem cell populations

Developing a New Two-Step Protocol to Generate Functional Hepatocytes from Wharton’s Jelly-Derived Mesenchymal Stem Cells under Hypoxic Condition

The shortage of donor livers and hepatocytes is a major limitation of liver transplantation. Thus, generation of hepatocyte-like cells may provide alternative choice for therapeutic applications. In this study, we developed a new method to establish hepatocytes from Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) cell lines named WJMSCs-SUT1 and WJMSCs-SUT2 under hypoxic condition. This new method could rapidly drive both WJ-MSCs cell lines into hepatic lineage within 18 days. The achievement of hepatogenic differentiation was confirmed by the characterization of both phenotypes and functions. More than 80% MSCs-derived hepatocyte-like cells (MSCDHCs) achieved functional hepatocytes including hepatic marker expressions both at gene and protein levels, glycogen storage, low-density lipoprotein uptake, urea production, and albumin secretion. This study highlights the establishment of new hepatogenic induction protocol under hypoxic condition in order to mimic hypoxic microenvironment in typical cell physiology. In conclusion, we present a simple, high-efficiency, and time saving protocol for the generation of functional hepatocyte-like cells from WJ-MSCs in hypoxic condition. The achievement of this method may overcome the limitation of donor hepatocytes and provides a new avenue for therapeutic value in cell-based therapy for life-threatening liver diseases, regenerative medicine, toxicity testing for pharmacological drug screening, and other medical related applications.

Generation of hepatocyte-like cells from human pluripotent stem cells for drug screening

Generation of hepatocyte-like cells from human pluripotent stem cells for drug screening

High yield generation of hepatocyte like cells from adipose derived stem cells

Mesenchyma stem cells (MSCs) are multipotent cells. They are capable of transforming into several mesoderm lineages. They can be separated from different tissues, such as bone marrow, umbilical cord blood, peripheral blood, and adipose tissue. Stem cell based therapies for the repair and regeneration of individual tissues and organs suggest a paradigm shift which may supply optional therapeutic solutions for a number of diseases. Embryonic and induced pluripotent stem cells have hypothetically been beneficial, although there are various restrictions. Bone marrow-derived mesenchyma stem cells when compared to adipose-derived stem cells (ADSCs), they were obviously capable of differentiating into cells and tissues with mesodermal origin. Adipose tissue is prevalent and easily available in large quantities. The intent of this investigation was the isolation of mesenchymal stem cells from rat adipose tissue, the potential of MSCs in differentiating into osteogenic and adipogenic lineages, describe the ADSCs surface expression of the classic markers by using flow cytometric analysis, and their transformation to hepatocyte like cells. Immunocytochemical analysis displayed that ADSCs express albumin and α-fetoprotein during differentiation. Knowledge of these cells may also demonstrate many benefits in cell-based therapies for tissue repair, regeneration, or tissue engineering. Key words: Hepatocyte-like cells, mesenchymal stem cells, cell therapy and adipose derived stem cell.

Efficient Generation of Hepatoblasts From Human ES Cells and iPS Cells by Transient Overexpression of Homeobox Gene HEX

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed α-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.

The generation of hepatocytes from mesenchymal stem cells and engraftment into murine liver

Donor organ shortage is still the major obstacle for the clinical application of hepatocyte transplantation in the treatment of liver diseases. However, generation of hepatocyte-like cells from mesenchymal stem cells (MSCs) has become a real alternative to the isolation of primary hepatocytes. MSCs are extracted from the tissue by collagenase digestion and enriched by their capacity to grow on plastic surfaces. Enriched cells display distinct mesenchymal surface markers and are capable of multiple lineage differentiation. In the presence of specific growth conditions, the cells adopt functional features of differentiated hepatocytes. After orthotopic transplantation, differentiated human stem cells engraft in the host liver parenchyma of immunocompromised mice. This protocol describes the in vitro differentiation of stem cells from human bone marrow and their transplantation into livers of immunodeficient mice. The cell culture procedures take about 4-5 weeks, and cells engrafted in the mouse liver may be detected 2-3 months after transplantation.

Stem cells for liver repopulation

The capacity of the liver to regenerate and maintain a constant size despite injury is unique. However, the exact mechanisms are not completely clear. Cell transplantation has been proposed as an alternative treatment of liver diseases. Recent progress has been reported on the generation of stem/progenitor cells that may differentiate toward the hepatic lineage. However, it is currently difficult to determine which of the stem/progenitor cell populations are the best for therapy of a given disease. The limited access to donor human hepatocytes has led to a great interest in the generation of hepatocyte-like cells. Several potential cell sources have been identified. However, general standardization of the methods to evaluate these cells is particularly important for the promise of stem/progenitor-derived hepatocyte-based therapies. Moreover, innovations aimed at improving hepatocyte delivery, survival, and engraftment have recently opened the field of organ engineering that may improve liver repopulation. Here we review current evidence reported from the perspective of potential clinical applications of different hepatic cell sources with repopulation capacities and the future perspectives and tools that can facilitate the translation of laboratory work into clinical success.