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.

Concentration-dependent toxicogenomic changes of silver nanoparticles in hepatocyte-like cells derived from human induced pluripotent stem cells.

The application of silver nanoparticles (AgNPs) in consumer products has been increasing rapidly over the past decades. Therefore, in vitro models capable of accurately predicting the toxicity of AgNPs are much needed. Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) represent an attractive alternative in vitro hepatotoxicity model. Yet, the use of iPSC-derived HLCs (iPSC-HLCs) for the study of nanoparticle toxicity has not been reported so far. In the present study, transcriptomic changes induced by varying concentrations (5-25 µg/ml) of AgNPs were characterized in iPSC-HLCs after 24-h exposure. AgNPs caused concentration-dependent gene expression changes in iPSC-HLCs. At all the concentrations, members of the metallothionein (MT) and the heat shock protein (HSP) families were the dominating upregulated genes, suggesting that exposure to AgNPs induced oxidative stresses in iPSC-HLCs and as a result elicited cellular protective responses in the cells. Functional analysis showed that the differentially expressed genes (DEGs) were majorly involved in the biological processes of metabolism, response to stress, and cell organization and biogenesis. Ingenuity Pathway Analysis revealed that cancer was at the top of diseases and disorders associated with the DEGs at all concentrations. These results were in accordance with those reported previously on hepatoma cell lines and primary hepatocytes. Considering the advantages iPSC-HLCs have over other liver cell models in terms of unlimited supply, consistency in quality, sustainability of function in long-term culture, and, more importantly, affordability of donor specificity, the results of the current study suggest that iPSC-HLCs may serve as a better in vitro model for liver nanotoxicology.

In vitro percutaneous penetration of silver nanoparticles in pig and human skin.

In this study, the effects of surface charge, dose, and cosmetic vehicle on the penetration of silver nanoparticles (AgNPs) into pig and human skin were compared. AgNPs (20 nm) with varying surface-charges (polyethylene glycol (PEG; neutral), citrate (CIT; negative), and branched polyethylenimine (bPEI; positive) were dosed onto skin in in vitro diffusion cells using an aqueous solution and an oil-in-water emulsion formulation. Samples were analyzed by inductively coupled plasma mass spectroscopy (ICP-MS) and transmission electron microscope (TEM) to assess AgNP skin penetration. The results showed that neutral and positive AgNPs penetrate human skin when applied in a high dose aqueous solution and less with the emulsion vehicle. A mass balance percutaneous penetration study in human skin found the majority of AgNPs were washed from the skin or remained mostly in the stratum corneum (3.4% of the applied dose for AgbPEI and 1.7% for AgPEG). Very little silver was found in the epidermis (1.2% AgbPEI and 0.3% AgPEG) and dermis (0.1% AgbPEI and none detected for AgPEG). These results indicate low dermal penetration of AgNPs that is not greatly affected by surface coating charge. The results will facilitate dermal exposure assessments by better understanding how nanoparticle properties affect skin absorption of nanoparticles found in personal care products.

Toxicity of nano- and ionic silver to embryonic stem cells: a comparative toxicogenomic study.

BACKGROUND: The widespread application of silver nanoparticles (AgNPs) and silver-containing products has raised public safety concerns about their adverse effects on human health and the environment. To date, in vitro toxic effects of AgNPs and ionic silver (Ag+) on many somatic cell types are well established. However, no studies have been conducted hitherto to evaluate their effect on cellular transcriptome in embryonic stem cells (ESCs). RESULTS: The present study characterized transcriptomic changes induced by 5.0 µg/ml AgNPs during spontaneous differentiation of mouse ESCs, and compared them to those induced by Ag+ under identical conditions. After 24 h exposure, 101 differentially expressed genes (DEGs) were identified in AgNP-treated cells, whereas 400 genes responded to Ag+. Despite the large differences in the numbers of DEGs, functional annotation and pathway analysis of the regulated genes revealed overall similarities between AgNPs and Ag+. In both cases, most of the functions and pathways impacted fell into two major categories, embryonic development and metabolism. Nevertheless, a number of canonical pathways related to cancer were found for Ag+ but not for AgNPs. Conversely, it was noted that several members of the heat shock protein and the metallothionein families were upregulated by AgNPs but not Ag+, suggesting specific oxidative stress effect of AgNPs in ESCs. The effects of AgNPs on oxidative stress and downstream apoptosis were subsequently confirmed by flow cytometry analysis. CONCLUSIONS: Taken together, the results presented in the current study demonstrate that both AgNPs and Ag+ caused transcriptomic changes that could potentially exert an adverse effect on development. Although transcriptomic responses to AgNPs and Ag+ were substantially similar, AgNPs exerted specific effects on ESCs due to their nanosized particulate form.

Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells.

Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment.

Toxicogenomic responses of human liver HepG2 cells to silver nanoparticles.

The increased use of silver nanoparticles (AgNPs) in foods and cosmetics has raised public safety concerns. However, only limited knowledge exists on the effect of AgNPs on the cellular transcriptome. This study evaluated global gene expression profiles of human liver HepG2 cells exposed to 20 and 50 nm AgNPs for 4 and 24 h at 2.5 µg ml(-1) . Exposure to 20 nm AgNPs resulted in 811 altered genes after 4 h, but much less after 24 h. Exposure to 50 nm AgNPs showed minimal altered genes at both exposure times. The HepG2 cells responded to the toxic insult of AgNPs by transiently upregulating stress response genes such as metallothioneins and heat shock proteins. Functional analysis of the altered genes showed more than 20 major biological processes were affected, of which metabolism, development, cell differentiation and cell death were the most dominant categories. Several cellular pathways were also impacted by AgNP exposure, including the p53 signaling pathway and the NRF2-mediated oxidative stress response pathway, which may lead to increased oxidative stress and DNA damage in the cell and potentially result in genotoxicity and carcinogenicity. Together, these results indicate that HepG2 cells underwent a multitude of cellular processes in response to the toxic insult of AgNP exposure, and suggest that toxicogenomic characterization of human HepG2 cells could serve as an alternative model for assessing toxicities of NPs.

Comparative genotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by a flow cytometric in vitro micronucleus assay.

Two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, and flow cytometry techniques were evaluated as tools for rapid screening of potential genotoxicity of food-related nanosilver. Comparative genotoxic potential of 20 nm silver was evaluated in HepG2 and Caco2 cell cultures by a flow cytometric-based in vitro micronucleus assay. The nanosilver, characterized by the dynamic light scattering, transmission electron microscopy and inductively coupled plasma-mass spectrometry analysis, showed no agglomeration of the silver nanoparticles. The inductively coupled plasma-mass spectrometry and transmission electron microscopy analysis demonstrated the uptake of 20 nm silver by both cell types. The 20 nm silver exposure of HepG2 cells increased the concentration-dependent micronucleus formation sevenfold at 10 µg ml(-1) concentration in attached cell conditions and 1.3-fold in cell suspension conditions compared to the vehicle controls. However, compared to the vehicle controls, the 20 nm silver exposure of Caco2 cells increased the micronucleus formation 1.2-fold at a concentration of 10 µg ml(-1) both in the attached cell conditions as well as in the cell suspension conditions. Our results of flow cytometric in vitro micronucleus assay appear to suggest that the HepG2 cells are more susceptible to the nanosilver-induced micronucleus formation than the Caco2 cells compared to the vehicle controls. However, our results also suggest that the widely used in vitro models, HepG2 and Caco2 cells and the flow cytometric in vitro micronucleus assay are valuable tools for the rapid screening of genotoxic potential of nanosilver and deserve more careful evaluation.

Comparative cytotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells in culture.

The use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics has increased significantly owing to their antibacterial and antifungal properties. As a consequence, the need for validated rapid screening methods to assess their toxicity is necessary to ensure consumer safety. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential cytotoxicity of food- and cosmetic-related nanoparticles. The two cell culture models were utilized to compare the potential cytotoxicity of 20-nm silver. The average size of the silver nanoparticle determined by our transmission electron microscopy (TEM) analysis was 20.4 nm. The dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The concentration of the 20-nm silver solution determined by our inductively coupled plasma-mass spectrometry (ICP-MS) analysis was 0.962 mg ml(-1) . Our ICP-MS and TEM analysis demonstrated the uptake of 20-nm silver by both HepG2 and Caco2 cells. Cytotoxicity, determined by the Alamar Blue reduction assay, was evaluated in the nanosilver concentration range of 0.1 to 20 µg ml(-1) . Significant concentration-dependent cytotoxicity of the nanosilver in HepG2 cells was observed in the concentration range of 1 to 20 µg ml(-1) and at a higher concentration range of 10 to 20 µg ml(-1) in Caco2 cells compared with the vehicle control. A concentration-dependent decrease in dsDNA content was observed in both cell types exposed to nanosilver but not controls, suggesting an increase in DNA damage. The DNA damage was observed in the concentration range of 1 to 20 µg ml(-1) . Nanosilver-exposed HepG2 and Caco2 cells showed no cellular oxidative stress, determined by the dichlorofluorescein assay, compared with the vehicle control in the concentration range used in this study. A concentration-dependent decrease in mitochondria membrane potential in both nanosilver exposed cell types suggested increased mitochondria injury compared with the vehicle control. The mitochondrial injury in HepG2 cells was significant in the concentration range of 1 to 20 µg ml(-1) , but in Caco2 cells it was significant at a higher concentration range of 10 to 20 µg ml(-1) . These results indicated that HepG2 cells were more sensitive to nanosilver exposure than Caco2 cells. It is generally believed that cellular oxidative stress induces cytotoxicity of nanoparticles. However, in this study we did not detect any nanosilver-induced oxidative stress in either cell type at the concentration range used in this study. Our results suggest that cellular oxidative stress did not play a major role in the observed cytotoxicity of nanosilver in HepG2 and Caco2 cells and that a different mechanism of nanosilver-induced mitochondrial injury leads to the cytotoxicity. The HepG2 and Caco2 cells used this study appear to be targets for silver nanoparticles. The results of this study suggest that the differences in the mechanisms of toxicity induced by nanosilver may be largely as a consequence of the type of cells used. This differential rather than universal response of different cell types exposed to nanoparticles may play an important role in the mechanism of their toxicity. In summary, the results of this study indicate that the widely used in vitro models, HepG2 and Caco2 cells in culture, are excellent systems for screening cytotoxicity of silver nanoparticles. These long established cell culture models and simple assays used in this study can provide useful toxicity and mechanistic information that can help to better inform safety assessments of food- and cosmetic-related silver nanoparticles.

Comparative genotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by fluorescent microscopy of cytochalasin B-blocked micronucleus formation.

As a consequence of the increased use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics to prevent fungal and bacterial growth, there is a need for validated rapid screening methods to assess the safety of nanoparticle exposure. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential genotoxicity of 20-nm nanosilver. The average silver nanoparticle size as determined by transmission electron microscopy (TEM) was 20.4 nm. Dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The silver concentration in a 20-nm nanosilver solution determined by the inductively coupled plasma-mass spectrometry (ICP-MS) analysis was 0.962 mg ml(-1) . Analysis by ICP-MS and TEM demonstrated the uptake of 20-nm silver by both HepG2 and Caco2 cells. Genotoxicity was determined by the cytochalasin B-blocked micronucleus assay with acridine orange staining and fluorescence microscopy. Concentration- and time-dependent increases in the frequency of binucleated cells with micronuclei induced by the nanosilver was observed in the concentration range of 0.5 to 15 µg ml(-1) in both HepG2 and Caco2 cells compared with the control. Our results indicated that HepG2 cells were more sensitive than Caco2 cells in terms of micronuclei formation induced by nanosilver exposure. In summary, the results of this study indicate that the widely used in vitro models, HepG2 and Caco2 cells in culture, represent potential screening models for prediction of genotoxicity of silver nanoparticles by in vitro micronucleus assay.

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.

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.

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.

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.

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.

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.

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.

Hepatocyte-like cells derived from human induced pluripotent stem cells using small molecules: implications of a transcriptomic study.

BACKGROUND: Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) hold great promise in toxicological applications as well as in regenerative medicine. Previous efforts on hepatocyte differentiation have mostly relied on the use of growth factors (GFs) to recapitulate developmental signals under in vitro conditions. Recently, the use of small molecules (SMs) has emerged as an attractive tool to induce cell fate transition due to its superiority in terms of both quality and cost. However, HLCs derived using SMs have not been well characterized, especially on the transcriptome level. METHODS: HLCs were differentiated from human iPSCs using a protocol that only involves SMs and characterized by transcriptomic analysis using whole genome microarrays. RESULTS: HLCs derived using the SM protocol (HLC_SM) displayed specific hepatic marker expression and demonstrated key hepatic functions. Transcriptomic analysis of the SM-driven differentiation defined a hepatocyte differentiation track and characterized the expression of some key marker genes in major stages of hepatocyte differentiation. In addition, HLC_SM were scored with CellNet, a bioinformatics tool quantifying how closely engineered cell populations resemble their target cell type, and compared to primary human hepatocytes (PHHs), adult liver tissue, fetal liver tissue, HLCs differentiated using GFs (HLC_GF), and commercially available HLCs. Similar to HLC_GF, HLC_SM displayed a mixed phenotype of fetal and adult hepatocytes and had relatively low expression of metabolic enzymes, transporters, and nuclear receptors compared to PHHs. Finally, the differentially expressed genes in HLC_SM compared to HLC_GF and to PHHs were analyzed to identify pathways and upstream transcription regulators which could potentially be manipulated to improve the differentiation of HLCs. CONCLUSIONS: Overall, the present study demonstrated the usefulness of the SM-based hepatocyte differentiation method, offered new insights into the molecular basis of hepatogenesis and associated gene regulation, and suggested ways for further improvements in hepatocyte differentiation in order to obtain more mature HLCs that could be used in toxicological studies.

A flow cytometric analysis of macrophage- nanoparticle interactions in vitro: induction of altered Toll-like receptor expression.

BACKGROUND: Nanoparticles exhibit unique physiochemical characteristics that provide the basis for their utilization. The diversity of potential and actual applications compels a thorough understanding regarding the consequences of their containment within the cellular environment. PURPOSE: This paper presents a flow cytometric examination of the biologic effects associated with the internalization of citrate-buffered silver (Ag) nanoparticles (NP) by the murine macrophage cell line, RAW264.7. MATERIALS AND METHODS: Cells were cultured with varying concentrations of citrate-buffered Ag nanoparticle and analyzed for changes in cellular volume, fluorescence emissions, and surface receptor expression. RESULTS: Notable changes in side scatter (SSC) signal occurred following the phagocytosis of citrate-buffered Ag NP representative of the 10 nm, 50 nm, and 100 nm particle size by cultured RAW 264.7 cells. A characteristic associated with the internalization of all the citrated Ag NP sizes tested, was the detection of emitted infra-red and near-infrared wavelength emissions. This characteristic consistently permitted the detection of 10 nm, 50 nm, and 100 nm Ag NP particles internalized within the RAW cells by flow cytometry. A functional distinction between monocyte subsets within the RAW 264.7 cell line was noted as Ag NP are taken up by the F4/80+ subset of cells within the culture. Further, the internalization of Ag NP by the cells resulted in an increased cell surface expression of the Toll-like receptor (TLR) 3, but not TLR4. CONCLUSION: Taken together, these results implicate the more mature macrophage in the ingestion of Ag NP; and an influence upon at least one of the Toll receptors present in macrophages following exposure to Ag NP. Further, our flow cytometric approach presents a potentially viable detection method for the identification of occult Ag NP material using an indicator cell line.

A Rapid and Highly Efficient Method for the Isolation, Purification, and Passaging of Human-Induced Pluripotent Stem Cells.

Human-induced pluripotent stem cells (iPSCs) hold considerable promise for future biomedical applications. However, the generation, isolation, and establishment of an iPSC line still presents many challenges. In this study, we describe a simple yet highly efficient two-step method for the isolation, purification, and passaging of human iPSC lines that utilizes commercially available reagents. The first step adapts iPSCs to single cell culture and passage, 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. Using this method, we were able to purify iPSCs from cell preparations containing differentiated or unreprogrammed cells, and even to isolate iPSC lines directly from derivation plates. The iPSC lines generated by this method maintained their pluripotency and genomic stability, as demonstrated by trilineage differentiation and karyotype analysis. The method presented here could be adopted for high-throughput isolation and expansion of iPSC lines and facilitate the widespread use of iPSCs in future applications.