Assessment of the effects of cannabidiol and a CBD-rich hemp extract in Caenorhabditis elegans.

Consumer use of cannabidiol (CBD) is growing, but there are still data gaps regarding its possible adverse effects on reproduction and development. Multiple pathways and signaling cascades involved in organismal development and neuronal function, including endocannabinoid synthesis and signaling systems, are well conserved across phyla, suggesting that Caenorhabditis elegans can model the in vivo effects of exogenous cannabinoids. The effects in C. elegans on oxidative stress response (OxStrR), developmental timing, juvenile and adult spontaneous locomotor activity, reproductive output, and organismal CBD concentrations were assessed after exposure to purified CBD or a hemp extract suspended in 0.5% sesame oil emulsions. In C. elegans, this emulsion vehicle is equivalent to a high-fat diet (HFD). As in mammals, HFD was associated with oxidative-stress-related gene expression in C. elegans adults. CBD reduced HFD-induced OxStrR in transgenic adults and counteracted the hypoactivity observed in HFD-exposed wild-type adults. In C. elegans exposed to CBD from the onset of feeding, delays in later milestone acquisition were irreversible, while later juvenile locomotor activity effects were reversible after the removal of CBD exposure. CBD-induced reductions in mean juvenile population body size were cumulative when chronic exposures were initiated at parental reproductive maturity. Purified CBD was slightly more toxic than matched concentrations of CBD in hemp extract for all tested endpoints, and both were more toxic to juveniles than to adults. Dosimetry indicated that all adverse effect levels observed in C. elegans far exceeded recommended CBD dosages for humans.

Strengths and limitations of the worm development and activity test (wDAT) as a chemical screening tool for developmental hazards.

The worm Development and Activity Test (wDAT) measures C. elegans developmental milestone acquisition timing and stage-specific spontaneous locomotor activity (SLA). Previously, the wDAT identified developmental delays and SLA level changes in C. elegans with mammalian developmental toxicants arsenic, lead, and mercury. 5-fluorouracil (5FU), cyclophosphamide (CP), hydroxyurea (HU), and ribavirin (RV) are teratogens that also induce growth retardation in developing mammals. In at least some studies on each of these chemicals, fetal weight reductions were seen at mammalian exposures below those that had teratogenic effects, suggesting that screening for developmental delay in a small alternative whole-animal model could act as a general toxicity endpoint to identify chemicals for further testing for more specific adverse developmental outcomes. Consistent with mammalian developmental effects, 5FU, HU, and RV were associated with developmental delays with the wDAT. Exposures associated with developmental delay induced hypoactivity with 5FU and HU, but slight hyperactivity with RV. CP is a prodrug that requires bioactivation by cytochrome P450s for both therapeutic and toxic effects. CP tests as a false negative in several in vitro assays, and it was also a false negative with the wDAT. These results suggest that the wDAT has the potential to identify some developmental toxicants, and that a positive wDAT result with an unknown may warrant further testing in mammals. Further assessment with larger panels of positive and negative controls will help qualify the applicability and utility of this C. elegans wDAT assay within toxicity test batteries or weight of evidence approaches for developmental toxicity assessment.

Effect of cannabidiol and hemp extract on viability and function of hepatocytes derived from human induced pluripotent stem cells.

Since the passage of the 2018 Agriculture Improvement Act (2018 Farm Bill), the number of products containing cannabis-derived compounds available to consumers have rapidly increased. Potential effects on liver function as a result from consumption of products containing cannabidiol (CBD), including hemp extracts, have been observed but the mechanisms for the effects are not fully understood. In this study, hepatocytes derived from human induced pluripotent stem cells (iPSCs) were used to evaluate potential hepatic effects of CBD and hemp extract at exposure concentrations ranging from 0.1 to 30 µM. Despite that a significant reduction in cell viability occurred only in the 30 µM group for both CBD and hemp extract, significant changes to cytochrome P450 activity, mitochondrial membrane potential, and lipid accumulation occurred within the concentration range of 0.1-3 µM for both CBD and hemp extract. Albumin and urea production, caspase 3/7 activity, and intracellular glutathione were significantly affected within the concentration range of 3-30 µM by CBD or hemp extract. These findings indicate that CBD and hemp extract can alter hepatic function and metabolism. The current study contributes data to help inform the evaluation of potential hepatotoxic effects of products containing cannabis-derived compounds.

Comparison on the mechanism and potency of hepatotoxicity among hemp extract and its four major constituent cannabinoids.

Cannabidiol (CBD) has been reported to induce hepatotoxicity in clinical trials and research studies; however, little is known about the safety of other nonintoxicating cannabinoids. New approach methodologies (NAMs) based on bioinformatic analysis of high-throughput transcriptomic data are gaining increasing importance in risk assessment and regulatory decision-making of data-poor chemicals. In the current study, we conducted a concentration response transcriptomic analysis of hemp extract and its four major constituent cannabinoids [CBD, cannabichromene (CBC), cannabigerol (CBG), and cannabinol (CBN)] in hepatocytes derived from human induced pluripotent stem cells (iPSCs). Each compound impacted a distinctive combination of biological functions and pathways. However, all the cannabinoids impaired liver metabolism and caused oxidative stress in the cells. Benchmark concentration (BMC) analysis showed potencies in transcriptional activity of the cannabinoids were in the order of CBN > CBD > CBC > CBG, consistent with the order of their cytotoxicity IC50 values. Patterns of transcriptomic changes induced by hemp extract and its median overall BMC were very similar to CBD but differed significantly from other cannabinoids, suggesting that potential adverse effects of hemp extract were largely due to its major constituent CBD. Lastly, transcriptomic point-of-departure (tPoD) values were determined for each of the compounds, with the value for CBD (0.106 µM) being concordant with a previously reported one derived from apical endpoints of clinical and animal studies. Taken together, the current study demonstrates the potential utility of transcriptomic BMC analysis as a NAM for hazard assessment of data-poor chemicals, improves our understanding of the possible health effects of hemp extract and its constituent cannabinoids, and provides important tPoD data that could contribute to inform human safety assessment of these cannabinoid compounds.

Hepatotoxicity of silver nanoparticles: Benchmark concentration modeling of an in vitro transcriptomics study in human iPSC-derived hepatocytes.

Despite two decades of research on silver nanoparticle (AgNP) toxicity, a safe threshold for exposure has not yet been established, albeit being critically needed for risk assessment and regulatory decision-making. Traditionally, a point-of-departure (PoD) value is derived from dose response of apical endpoints in animal studies using either the no-observed-adverse-effect level (NOAEL) approach, or benchmark dose (BMD) modeling. To develop new approach methodologies (NAMs) to inform human risk assessment of AgNPs, we conducted a concentration response modeling of the transcriptomic changes in hepatocytes derived from human induced pluripotent stem cells (iPSCs) after being exposed to a wide range concentration (0.01-25 µg/ml) of AgNPs for 24 h. A plausible transcriptomic PoD of 0.21 µg/ml was derived for a pathway related to the mode-of-action (MOA) of AgNPs, and a more conservative PoD of 0.10 µg/ml for a gene ontology (GO) term not apparently associated with the MOA of AgNPs. A reference dose (RfD) could be calculated from either of the PoDs as a safe threshold for AgNP exposure. The current study illustrates the usefulness of in vitro transcriptomic concentration response study using human cells as a NAM for toxicity study of chemicals that lack adequate toxicity data to inform human risk assessment.

Oxidative DNA damage contributes to usnic acid-induced toxicity in human induced pluripotent stem cell-derived hepatocytes.

Dietary supplements containing usnic acid have been increasingly marketed for weight loss over the past decades, even though incidences of severe hepatotoxicity and acute liver failure due to their overuse have been reported. To date, the toxic mechanism of usnic acid-induced liver injury at the molecular level still remains to be fully elucidated. Here, we conducted a transcriptomic study on usnic acid using a novel in vitro hepatotoxicity model employing human induced pluripotent stem cell (iPSC)-derived hepatocytes. Treatment with 20 µM usnic acid for 24 h caused 4272 differentially expressed genes (DEGs) in the cells. Ingenuity Pathway Analysis (IPA) based on the DEGs and gene set enrichment analysis (GSEA) using the whole transcriptome expression data concordantly revealed several signaling pathways and biological processes that, when taken together, suggest that usnic acid caused oxidative stress and DNA damage in the cells, which further led to cell cycle arrest and eventually resulted in cell death through apoptosis. These transcriptomic findings were subsequently corroborated by a variety of cellular assays, including reactive oxygen species (ROS) generation and glutathione (GSH) depletion, DNA damage (pH2AX detection and 8-hydroxy-2'-deoxyguanosine [8-OH-dg] assay), cell cycle analysis, and caspase 3/7 activity. Collectively, the results of the current study accord with previous in vivo and in vitro findings, provide further evidence that oxidative stress-caused DNA damage contributes to usnic acid-induced hepatotoxicity, shed new light on molecular mechanisms of usnic acid-induced hepatotoxicity, and demonstrate the usefulness of iPSC-derived hepatocytes as an in vitro model for hepatotoxicity testing and prediction.

Toxicological applications of human induced pluripotent stem cell-derived hepatocyte-like cells: an updated review.

Variability in supply, paucity of donors and cellular instability under in vitro conditions have limited the application of primary human hepatocytes (PHHs) to hepatotoxicity testing. Therefore, alternative sources have been sought for functional liver cells. Many of the earlier in vitro hepatotoxicity studies were carried out using hepatoma-derived cell lines. These cell lines have overcome some of the limitations of PHHs with regard to phenotypic stability and availability; however, they suffer from their own inherent limitations, such as the lack of drug-metabolizing functionality, which renders them inadequate for situations where toxic metabolite formation of the parent drug occurs. In the last decade we have witnessed a burgeoning interest of the research community in using hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) as in vitro hepatotoxicity models. HLCs offer the perspective of a defined and renewable supply of functional hepatocytes; more importantly, HLCs maintain their original donor genotype and afford donor diversity, thus opening new avenues to patient-specific toxicity testing. In this review, we first introduce various in vitro hepatotoxicity models, then focus on HLCs and their application in hepatotoxicity studies, and finally offer some perspectives on future developments of the field.

The worm Adult Activity Test (wAAT): A de novo mathematical model for detecting acute chemical effects in Caenorhabditis elegans.

We have adapted a semiautomated method for tracking Caenorhabditis elegans spontaneous locomotor activity into a quantifiable assay by developing a sophisticated method for analyzing the time course of measured activity. The 16-h worm Adult Activity Test (wAAT) can be used to measure C. elegans activity levels for efficient screening for pharmacological and toxicity-induced effects. As with any apical endpoint assay, the wAAT is mode of action agnostic, allowing for detection of effects from a broad spectrum of response pathways. With caffeine as a model mild stimulant, the wAAT showed transient hyperactivity followed by reversion to baseline. Mercury chloride (HgCl2 ) produced an early dose-response hyperactivity phase followed by pronounced hypoactivity, a behavior pattern we have termed a toxicant "escape response." Methylmercury chloride (meHgCl) produced a similar pattern to HgCl2 , but at much lower concentrations, a weaker hyperactivity response, and more pronounced hypoactivity. Sodium arsenite (NaAsO2 ) and dimethylarsinic acid (DMA) induced hypoactivity at high concentrations. Acute toxicity, as measured by hypoactivity in C. elegans adults, was ranked: meHgCl > HgCl2 > NaAsO2 = DMA. Caffeine was not toxic with the wAAT at tested concentrations. Methods for conducting the wAAT are described, along with instructions for preparing C. elegans Habitation Medium, a liquid nutrient medium that allows for developmental timing equivalent to that found with C. elegans grown on agar with OP50 Escherichia coli feeder cultures. A de novo mathematical parametric model for adult C. elegans activity and the application of this model in ranking exposure toxicity are presented.

Reproductive-Toxicity-Related Endpoints in C. elegans Are Consistent with Reduced Concern for Dimethylarsinic Acid Exposure Relative to Inorganic Arsenic.

Exposures to arsenic and mercury are known to pose significant threats to human health; however, the effects specific to organic vs. inorganic forms are not fully understood. Caenorhabditis elegans' (C. elegans) transparent cuticle, along with the conservation of key genetic pathways regulating developmental and reproductive toxicology (DART)-related processes such as germ stem cell renewal and differentiation, meiosis, and embryonic tissue differentiation and growth, support this model's potential to address the need for quicker and more dependable testing methods for DART hazard identification. Organic and inorganic forms of mercury and arsenic had different effects on reproductive-related endpoints in C. elegans, with methylmercury (meHgCl) having effects at lower concentrations than mercury chloride (HgCl2), and sodium arsenite (NaAsO2) having effects at lower concentrations than dimethylarsinic acid (DMA). Progeny to adult ratio changes and germline apoptosis were seen at concentrations that also affected gravid adult gross morphology. For both forms of arsenic tested, germline histone regulation was altered at concentrations below those that affected progeny/adult ratios, while concentrations for these two endpoints were similar for the mercury compounds. These C. elegans findings are consistent with corresponding mammalian data, where available, suggesting that small animal model test systems may help to fill critical data gaps by contributing to weight of evidence assessments.

Predicting binding between 55 cannabinoids and 4,799 biological targets by in silico methods.

Recently, there has been an increase in cannabis-derived products being marketed as foods, dietary supplements, and other consumer products. Cannabis contains over a hundred cannabinoids, many of which have unknown physiological effects. Since there are large numbers of cannabinoids, and many are not commercially available for in vitro testing, an in silico tool (Chemotargets Clarity software) was used to predict binding between 55 cannabinoids and 4,799 biological targets (enzymes, ion channels, receptors, and transporters). This tool relied on quantitative structure activity relationships (QSAR), structural similarity, and other approaches to predict binding. From this screening, 827 cannabinoid-target binding pairs were predicted, which included 143 unique targets. Many cannabinoids sharing core structures (cannabinoid "types") had similar binding profiles, whereas most cannabinoids containing carboxylic acid groups were similar without regards to their core structure. For some of the binding predictions (43), in vitro binding data were available, and they agreed well with in silico binding data (median fourfold difference in binding concentrations). Finally, clinical adverse effects associated with 22 predicted targets were identified from an online database (Clarivate Off-X), providing important insights on potential human health hazards. Overall, in silico biological target predictions are a rapid means to identify potential hazards due to cannabinoid-target interactions, and the data can be used to prioritize subsequent in vitro and in vivo testing.

Development and validation of a fit-for-purpose UHPLC-ESI-MS/MS method for the quantitation of cannabinoids in different matrices.

Several cannabinoids (cannabidivarin (CBDV), cannabigerol (CBG), cannabidiol (CBD), cannabinol (CBN) and cannabichromene (CBC)) and ethanol hemp extract are being used in primary human hepatocytes (PHH), Caenorhabditis elegans (C. elegans) and in vitro buccal membrane absorption models to elucidate their potential toxicological mechanisms, evaluate their oromucosal absorption, and to identify their metabolites. William's E medium, C. elegans habitation medium (CeHM), and HEPES-buffered hanks' balanced salt solution (HHBSS) are matrices used with these predictive test systems. Therefore, we developed and validated a sensitive fit-for-purpose ultra-high performance liquid chromatography-electrospray-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantitation of CBDV, CBG, CBD, CBN, and CBC in extracellular matrices used with these models for the first time. The separation of the analytes was performed on a Waters ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 × 100 mm) protected with a Waters ACQUITY UPLC BEH C18 guard column (130 Å, 1.7 µm, 2.1 × 5 mm). Positive electrospray ionization and multiple reaction monitoring (MRM) modes were used. Under the developed experimental conditions, good linearities were obtained over the concentration range of 0.025-40 µg/ml with coefficients of determination (R2) varying from 0.9953 to 0.9998. The intra-day precisions were between 0.5 and 9.6% with accuracies within ± 16.7%, and the inter-day precisions ranged from 0.6 to 13.1 % with accuracies within ± 13.7%. The method recoveries were between 85.8 and 105.1%. In addition, time-consuming sample preparation was avoided by applying a simple and efficient extraction procedure, which meets the need for potential large-scale routine analysis. The described method was successfully applied to quantitate the analytes in samples produced with different models as well as in ethanolic hemp extract.

Picamilon, a γ-aminobutyric acid (GABA) analogue and marketed nootropic, is inactive against 50 biological targets.

Picamilon is an analogue of the neurotransmitter γ-aminobutyric acid (GABA), which is marketed as a nootropic claiming to enhance cognition. There is a lack of in silico, in vitro and in vivo data on the safety of picamilon. Therefore, to ascertain potential physiological effects of picamilon, it was screened against 50 safety-related biological targets (receptors, ion channels, enzymes and transporters) by in silico and in vitro methods. Using two in silico tools, picamilon was not predicted to bind to the targets. Similarly, picamilon exhibited weak or no binding to the targets when measured in vitro at 10 µM. Overall, this data shows that picamilon, although structurally similar to other GABA analogues, has a different biological target binding profile. Picamilon's lack of binding to the 50 targets fills important data gaps among GABA analogues, a group of structurally related substances found in drugs and other consumer products.

Physiologically based pharmacokinetic modeling and simulation of cannabinoids in human plasma and tissues.

There has been an increased public interest in developing consumer products containing nonintoxicating cannabinoids, such as cannabidiol (CBD) and cannabigerol (CBG). At the present time, there is limited information available on the pharmacokinetics of cannabinoids in humans. Since pharmacokinetic profiles are important in understanding the pharmacological and toxicological effects at the target sites, physiologically based pharmacokinetic (PBPK) modeling was used to predict the plasma and tissue concentrations of 17 cannabinoids in humans. PBPK models were established using measured (in vitro) and predicted (in silico) physicochemical and pharmacokinetic properties, such as water solubility and effective human jejunal permeability. Initially, PBPK models were established for CBD and the model performance was evaluated using reported clinical data after intravenous and oral administration. PBPK models were then developed for 16 additional cannabinoids including CBG, and the plasma and tissue concentrations were predicted after 30 mg oral administration. The pharmacokinetic profiles of the 16 cannabinoids were similar to CBD, and the plasma concentration and time profiles of CBD agreed well with clinical data in the literature. Although low exposure was predicted in the plasma (maximum plasma concentrations < 15 nM), the predicted tissue concentrations, especially the liver (maximum liver concentrations 70-183 nM), were higher after oral administration of 30 mg cannabinoids. These predicted plasma and tissue concentrations could be used to guide further in vitro and in vivo testing.

A transcriptomic dataset comparing two methods of hepatocyte differentiation from human induced pluripotent stem cells.

A variety of methods have been reported for the differentiation of hepatocyte-like cells (HLCs) from human induced pluripotent stem cells (iPSCs) using various growth factors or small molecules. However, direct comparison of the differentiation efficiency and the quality of the final HLCs between different methods has rarely been reported. To fill this data gap, we compared two hepatocyte differentiation methods, termed Method 1 and Method 2, and published the major findings in a research article entitled "Phenotypical, functional and transcriptomic comparison of two modified methods of hepatocyte differentiation from human induced pluripotent stem cells" (Li et al., 2022). The current data article describes the transcriptomic dataset comparing the two methods. HLCs were collected at early maturation (day 17) and late maturation (day 21) stages of the differentiation and total RNA were isolated. Global gene expression profiling of the HLCs was conducted using Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Primary human hepatocytes (PHHs) were also included for comparison. The microarray dataset has been deposited in the Gene Expression Omnibus of the National Center for Biotechnology Information with accession number GSE187011. Detailed interpretation and discussion of the data can be found in the corresponding research article (Li et al., 2022). This dataset is useful in providing a molecular basis for the differences observed between the two differentiation methods, offering new insights into gene regulations in hepatogenesis in vitro, and suggesting ways to further improve hepatocyte differentiation in order to obtain more mature HLCs for biomedical applications.

Rapid and Highly Efficient Isolation and Purification of Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) hold great promise for biomedical applications. However, establishment of new iPSC lines still presents many challenges. Here we describe a simple yet highly efficient two-step protocol for the isolation and purification of human iPSC lines. The first step adapts iPSCs to single cell culture and passaging, promoting survival and self-renewal; the second step enables the isolation and purification of bona fide iPSCs from a mixed population using column-based positive selection of cells expressing pluripotency markers such as TRA-1-60. Both steps utilize commercially available reagents. Using this protocol, iPSCs can be purified from cell preparations containing differentiated or unreprogrammed cells, or even be isolated directly from reprogramming vessels. The protocol could be adopted for high throughput isolation and expansion of iPSC lines and facilitate the widespread use of iPSCs in future applications.

Homogeneous Differentiation of Functional Hepatocytes from Human Induced Pluripotent Stem Cells.

Hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (iPSCs) could provide an unlimited source of liver cells for regenerative medicine, disease modeling, drug screening, and toxicology studies. Here we describe a stepwise improved protocol that enables highly efficient, homogeneous, and reproducible differentiation of human iPSCs into functional hepatocytes through controlling all three stages of hepatocyte differentiation, starting from a single cell (non-colony) culture of iPSCs, through homogeneous definitive endoderm induction and highly efficient hepatic specification, and finally arriving at matured HLCs. The final population of cells exhibits morphology closely resembling that of primary human hepatocytes, and expresses specific hepatic markers as evidenced by immunocytochemical staining. More importantly, these HLCs demonstrate key functional characteristics of mature hepatocytes, including major serum protein (e.g., albumin, fibronectin, and alpha-1 antitrypsin) secretion, urea synthesis, glycogen storage, and inducible cytochrome P450 activity.

Assessment of the effects of organic vs. inorganic arsenic and mercury in Caenorhabditis elegans.

Exposures to mercury and arsenic are known to pose significant threats to human health. Effects specific to organic vs. inorganic forms of these toxic elements are less understood however, especially for organic dimethylarsinic acid (DMA), which has recently been detected in pups of rodent dams orally exposed to inorganic sodium (meta)arsenite (NaAsO2). Caenorhabditis elegans is a small animal alternative toxicity model. To fill data gaps on the effects of DMA relative to NaAsO2, C. elegans were exposed to these two compounds alongside more thoroughly researched inorganic mercury chloride (HgCl2) and organic methylmercury chloride (meHgCl). For timing of developmental milestone acquisition in C. elegans, meHgCl was 2 to 4-fold more toxic than HgCl2, and NaAsO2 was 20-fold more toxic than DMA, ranking the four compounds meHgCl > HgCl2 > NaAsO2 ≫ DMA for developmental toxicity. Methylmercury induced significant decreases in population locomotor activity levels in developing C. elegans. DMA was also associated with developmental hypoactivity, but at >100-fold higher concentrations than meHgCl. Transcriptional alterations in native genes were observed in wild type C. elegans adults exposed to concentrations equitoxic for developmental delay in juveniles. Both forms of arsenic induced genes involved in immune defense and oxidative stress response, while the two mercury species induced proportionally more genes involved in transcriptional regulation. A transgenic bioreporter for activation of conserved proteosome specific unfolded protein response was strongly activated by NaAsO2, but not DMA at tested concentrations. HgCl2 and meHgCl had opposite effects on a bioreporter for unfolded protein response in the endoplasmic reticulum. Presented experiments indicating low toxicity for DMA in C. elegans are consistent with human epidemiologic data correlating higher arsenic methylation capacity with resistance to arsenic toxicity. This work contributes to the understanding of the accuracy and fit-for-use categories for C. elegans toxicity screening and its usefulness to prioritize compounds of concern for further testing.

Phenotypical, functional and transcriptomic comparison of two modified methods of hepatocyte differentiation from human induced pluripotent stem cells.

Directed differentiation of human induced pluripotent stem cells (iPSCs) into hepatocytes could provide an unlimited source of liver cells, and therefore holds great promise for regenerative medicine, disease modeling, drug screening and toxicology studies. Various methods have been established during the past decade to differentiate human iPSCs into hepatocyte-like cells (HLCs) using growth factors and/or small molecules. However, direct comparison of the differentiation efficiency and the quality of the final HLCs between different methods has rarely been reported. In the current study, two hepatocyte differentiation methods were devised, termed Method 1 and 2, through modifying existing well-known hepatocyte differentiation strategies, and the resultant cells were compared phenotypically and functionally at different stages of hepatocyte differentiation. Compared to Method 1, higher differentiation efficiency and reproducibility were observed in Method 2, which generated highly homogeneous functional HLCs at the end of the differentiation process. The cells exhibited morphology closely resembling primary human hepatocytes and expressed high levels of hepatic protein markers. More importantly, these HLCs demonstrated several essential characteristics of mature hepatocytes, including major serum protein (albumin, fibronectin and α-1 antitrypsin) secretion, urea release, glycogen storage and inducible cytochrome P450 activity. Further transcriptomic comparison of the HLCs derived from the two methods identified 1,481 differentially expressed genes (DEGs); 290 Gene Ontology terms in the biological process category were enriched by these genes, which were further categorized into 34 functional classes. Pathway analysis of the DEGs identified several signaling pathways closely involved in hepatocyte differentiation of pluripotent stem cells, including 'signaling pathways regulating pluripotency of stem cells', 'Wnt signaling pathway', 'TGF-beta signaling pathway' and 'PI3K-Akt signaling pathway'. These results may provide a molecular basis for the differences observed between the two differentiation methods and suggest ways to further improve hepatocyte differentiation in order to obtain more mature HLCs for biomedical applications.

Generation of Human Induced Pluripotent Stem Cells Using Endothelial Progenitor Cells Derived from Umbilical Cord Blood and Adult Peripheral Blood.

Induced pluripotent stem cells (iPSCs) offer the potential to generate tissue cells with donor diversity therefore promising to have widespread applications in regenerative medicine, disease modeling, drug discovery, and toxicity testing. Several somatic cell types have been utilized, with varying efficiencies, as source cells for the reprogramming of iPSCs. Recently, it has been reported that endothelial progenitor cells (EPCs) derived from umbilical cord blood (CB) or adult peripheral blood (PB) afford a practical and efficient cellular substrate for iPSC generation, and possess several advantages over other cell types. In this chapter, we describe a protocol that covers all steps of reprogramming iPSCs from blood-derived EPCs, including (1) isolation of mononuclear cells (MNCs) from blood samples, (2) derivation of EPCs from MNCs, and (3) generation of iPSCs from EPCs. The final step of reprogramming EPCs into iPSCs is achieved through ectopic expression of four transcription factors, OCT4, KLF4, SOX2, and c-MYC, using self-replicative RNA (srRNA) technology.

Transcriptomic and proteomic responses of silver nanoparticles in hepatocyte-like cells derived from human induced pluripotent stem cells.

Silver nanoparticles (AgNPs) have been increasingly used in a variety of consumer products over the last decades. However, their potential adverse effects have not been fully understood. In a previous study, we characterized transcriptomic changes in human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) in response to AgNP exposure. Here, we report findings of a follow-up proteomic study that evaluated alternations at the protein level in the same cell after being exposed to 10 µg/ml AgNPs for 24 h. In total, 6287 proteins were identified across two groups of samples (n = 3). Among these proteins, 665 were found to be differentially regulated (fold change ≥1.25, p < 0.01) between the AgNP-treated group and the untreated control group, including 264 upregulated and 401 downregulated. Bioinformatics analysis of the proteomics data, in side-by-side comparison to the transcriptomics data, confirms and substantiates previous findings on AgNP-induced alterations in metabolism, oxidative stress, inflammation, and potential association with cancer. A mechanism of action was proposed based on these results. Collectively, the findings of the current proteomic study are consistent with those of the previous transcriptomic study and further demonstrate the usefulness of iPSC-derived HLCs as an in vitro model for liver nanotoxicology.