Size‐Dependent Toxicity of Gold Nanoparticles on Human Embryonic Stem Cells and Their Neural Derivatives

This study explores the use of human embryonic stem cells (hESCs) for assessing nanotoxicology, specifically, the effect of gold nanoparticles (AuNPs) of different core sizes (1.5, 4, and 14 nm) on the viability, pluripotency, neuronal differentiation, and DNA methylation of hESCs. The hESCs exposed to 1.5 nm thiolate‐capped AuNPs exhibit loss of cohesiveness and detachment suggesting ongoing cell death at concentrations as low as 0.1 μg mL^?1. The cells exposed to 1.5 nm AuNPs at this concentration do not form embryoid bodies but rather disintegrate into single cells within 48 h. Cell death caused by 1.5 nm AuNPs also occur in hESC‐derived neural progenitor cells. None of the other nanoparticles exhibit toxic effects on the hESCs at concentrations as high as 10 μg mL^?1 during a 19 d neural differentiation period. Thiolate‐capped 4 nm AuNPs at 10 μg mL^?1 cause a dramatic decrease in global DNA methylation (5 mC) and a corresponding increase in global DNA hydroxymethylation (5 hmC) of the hESC's DNA in only 24 h. This work identifies a type of AuNPs highly toxic to hESCs and demonstrates the potential of hESCs in predicting nanotoxicity and characterizing their ability to alter the DNA methylation and hydroxymethylation patterns in the cells.     

Role of toll-like receptors 3, 4 and 7 in cellular uptake and response to titanium dioxide nanoparticles

Innate immune response is believed to be among the earliest provisional cellular responses, and mediates the interactions between microbes and cells. Toll-like receptors (TLRs) are critical to these interactions. We hypothesize that TLRs also play an important role in interactions between nanoparticles (NPs) and cells, although little information has been reported concerning such an interaction. In this study, we investigated the role of TLR3, TLR4 and TLR7 in cellular uptake of titanium dioxide NP (TiO₂ NP) agglomerates and the resulting inflammatory responses to these NPs. Our data indicate that TLR4 is involved in the uptake of TiO₂ NPs and promotes the associated inflammatory responses. The data also suggest that TLR3, which has a subcellular location distinct from that of TLR4, inhibits the denaturation of cellular protein caused by TiO₂ NPs. In contrast, the unique cellular localization of TLR7 has middle-ground functional roles in cellular response after TiO₂ NP exposure. These findings are important for understanding the molecular interaction mechanisms between NPs and cells.     

Low doses of TiO2-polyethylene glycol nanoparticles stimulate proliferation of hepatocyte cells

This paper describes the effect of low concentrations of 100 nm polyethylene glycol-modified TiO₂ nanoparticles (TiO₂-PEG NPs) on HepG2 hepatocellular carcinoma cells. Proliferation of HepG2 cells increased significantly when the cells were exposed to low doses (<100 μg ml^–1) of TiO₂-PEG NPs. These results were further confirmed by cell counting experiments and cell cycle assays. Cellular uptake assays were performed to determine why HepG2 cells proliferate with low-dose exposure to TiO₂-PEG NPs. The results showed that exposure to lower doses of NPs led to less cellular uptake, which in turn decreased cytotoxicity. We therefore hypothesized that TiO₂-PEG NPs could affect the activity of hepatocyte growth factor receptors (HGFRs), which bind to hepatocyte growth factor and stimulate cell proliferation. The localization of HGFRs on the surface of the cell membrane was detected via immunofluorescence staining and confocal microscopy. The results showed that HGFRs aggregate after exposure to TiO₂-PEG NPs. In conclusion, our results indicate that TiO₂-PEG NPs have the potential to promote proliferation of HepG2 cells through HGFR aggregation and suggest that NPs not only exhibit cytotoxicity but also affect cellular responses.     

Penetration,distribution and brain toxicity of titanium nanoparticles in rodents' body: a review

Titanium dioxide (TiO₂) has been vastly used commercially, especially as white pigment in paints, colorants, plastics, coatings, cosmetics. Certain industrial uses TiO₂ in diameter <100 nm. There are three common exposure routes for TiO₂ : (i) inhalation exposure, (ii) exposure via gastrointestinal tract, (iii) dermal exposure. Inhalation and gastrointestinal exposure appear to be the most probable ways of exposure, although nanoparticle (NP) penetration is limited. However, the penetration rate may increase substantially when the tissue is impaired. When TiO₂ NPs migrate into the circulatory system, they can be distributed into all tissues including brain. In brain, TiO₂ lead to oxidative stress mediated by the microglia phagocytic cells which respond to TiO₂ NPs by the production and release of superoxide radicals that convert to multiple reactive oxygen species (ROS). The ROS production may also cause the damage of blood–brain barrier which then becomes more permeable for NPs. Moreover, several studies have showed neuron degradation and the impairment of spatial recognition memory and learning abilities in laboratory rodent exposed to TiO₂ NPs.Inspec keywords: nanoparticles, permeability, health hazards, molecular biophysics, biochemistry, biological tissues, toxicology, oxidation, brain, neurophysiology, blood, reviews, cosmetics, cellular biophysics, titanium compoundsOther keywords: rodents, white pigment, gastrointestinal tract, dermal exposure, gastrointestinal exposure, nanoparticle penetration, penetration rate, blood–brain barrier, titanium nanoparticles, brain toxicity, size 100.0 nm, TiO₂      

Toxicological Aspects of Long‐Term Treatment of Keratinocytes with ZnO and TiO2 Nanoparticles

Sunscreens containing ZnO and TiO₂ nanoparticles (NPs) are increasingly applied to skin over long time periods to reduce the risk of skin cancer. However, long‐term toxicological studies of NPs are very sparse. The in vitro toxicity of ZnO and TiO₂ NPs on keratinocytes over short‐ and long‐term applications is reported. The effects studied are intracellular formation of radicals, alterations in cell morphology, mitochondrial activity, and cell‐cycle distribution. Cellular response depends on the type of NP, concentration, and exposure time. ZnO NPs have more pronounced adverse effects on keratinocytes than TiO₂. TiO₂ has no effect on cell viability up to 100 μg mL^?1, whereas ZnO reduces viability above 15 μg mL^?1 after short‐term exposure. Prolonged exposure to ZnO NPs at 10 μg mL^?1 results in decreased mitochondrial activity, loss of normal cell morphology, and disturbances in cell‐cycle distribution. From this point of view TiO₂ has no harmful effect. More nanotubular intercellular structures are observed in keratinocytes exposed to either type of NP than in untreated cells. This observation may indicate cellular transformation from normal to tumor cells due to NP treatment. Transmission electron microscopy images show NPs in vesicles within the cell cytoplasm, particularly in early and late endosomes and amphisomes. Contrary to insoluble TiO₂, partially soluble ZnO stimulates generation of reactive oxygen species to swamp the cell redox defense system thus initiating the death processes, seen also in cell‐cycle distribution and fluorescence imaging. Long‐term exposure to NPs has adverse effects on human keratinocytes in vitro, which indicates a potential health risk.     

TiO2 Nanorod Array Constructed Nanotopography for Regulation of Mesenchymal Stem Cells Fate and the Realization of Location‐Committed Stem Cell Differentiation

As a physical cue for controlling the fate of stem cells, surface nanotopography has attracted much attention to improve the integration between implants and local host tissues and cells. A biocompatible surface TiO₂ nanorod array is proposed to regulate the fate of bone marrow derived mesenchymal stem cells (MSCs). TiO₂ substrates with different surface nanotopographies: a TiO₂ nanorod array and a polished TiO₂ ceramic are built by hydrothermal and sintering processes, respectively. The assessment of morphology, viability, gene expression, and protein characterization of the MSCs cultured on the different TiO₂ substrates proves that a TiO₂ nanorod array promotes the osteogenic differentiation of MSCs, while a TiO₂ ceramic with a smooth surface suppresses it. Periodically assembled TiO₂ nanorod array stripes on the smooth TiO₂ ceramic are constructed by a combination of microfabrication and a chemical synthesis process, which realizes the location‐committed osteogenic differentiation of MSCs. A route to control the differentiation of MSCs by a nanostructured surface, which can also control the location and direction of MSCs on the surface of biomaterials with micro‐nano scale surface engineering, is demonstrated.     

Toxicological impact of TiO2 nanoparticles on Eudrilus euginiae

The concern regarding the toxicological effects of nanoparticles (NPs) on the terrestrial environment is increasing. To avoid risks of exposure to these NPs in the environment, it is essential to develop an understanding of their reactivity, toxicity, and persistency. Due to the increased usage of nano‐titanium dioxide (TiO₂) in various industrial products, an exponential increase in exposure is expected, which would exacerbate concerns about its ecological risks. The present study is conducted to evaluate the size‐dependent effects of TiO₂ NPs on the soil, especially on earthworm (Eudrilus euginiae). To date, many studies have been reported on the impact of TiO₂ NPs on ecotoxicology. However, histotoxicology studies are sparse. This study serves to be the first report on the size‐dependent histotoxicological impact of nano‐TiO₂ on earthworms particularly, E. euginiae. This report presents an intensive overall view of the longer time ecotoxicological impact of TiO₂ nanomaterials on various biological parameters of earthworms at cellular levels. The results show that the survival and growth of adult earthworms are severely affected by the TiO₂ NPs in the soil, which substantiates the adverse effects of TiO₂ NPs on earthworms.Inspec keywords: nanobiotechnology, nanoparticles, titanium compounds, semiconductor materials, toxicology, zoology, soil, cellular biophysics, particle sizeOther keywords: toxicological impact, nanoparticles, Eudrilus euginiae, terrestrial environment, soil, earthworm, ecotoxicology, size‐dependent histotoxicological impact, nanomaterials, biological parameters, cellular levels, TiO₂      

Cytotoxicity of titanium dioxide nanoparticles differs in four liver cells from human and rat

Titanium dioxide (TiO₂) nanoparticles (NPs) are the important nanoscale components of composites. Although TiO₂ NPs and their related nanocomposites have been widely used in industrial and medical applications, the adverse effects of TiO₂ nanomaterials have not been well studied. Here, we investigated the cytotoxicity of TiO₂ NPs in vitro using four liver cell lines: human hepatocellular carcinoma cell line (SMMC-7721), human liver cell line (HL-7702), rat hepatocarcinoma cell line (CBRH-7919) and rat liver cell line (BRL-3A). We checked cell viability, cell morphology, and the levels of reactive oxygen species (ROS) and glutathione (GSH) after TiO₂ exposure at varying concentrations (0.1–100 μg/mL) and different exposure periods of time (12–48 h). Compared to the NP-free control, all four cell lines exposed to TiO₂ NPs showed cytotoxicity in a dosage-dependent and time-dependent manner, which was associated with the changes of cell viability and cell morphology, increased intercellular ROS levels, and decreased intracellular GSH levels. Further, we observed that carcinomatous liver cells and human liver cells exhibited more tolerance to TiO₂ NPs exposure for 24 h, compared to normal liver cells and rat liver cells, respectively. The results indicate that the in vitro cytotoxicity induced by NPs should be assessed with great caution before the use of nanocomposites and that there is a need to standardize the cytotoxicity testing procedure of nanoscale components in composites when using different cell lines.     

Cardiac and testicular alterations induced by acute exposure to titanium dioxide nanoparticles: Histopathological study

Titanium dioxide nanoparticles (TiO₂ NPs) have novel application and are used in many household application, nanomedicine, agriculture, industries and pharmaceutical products. These applications may be accompanied with potential risk in human health and the ecosystems. The current study was carried out to find out the acute damage that might be induced by TiO₂ NPs in the heart and testis. Three groups of Wistar albino rats (Rattus norvegicus) were subjected to a single dose TiO₂ NPs (126, 252, 378 mg/kg bw). Cardiac and testicular biopsies from each animal under study were handled for histological and histochemical examination. Rats exposed to TiO₂ NPs demonstrated the following cardiac alterations: myofibres wavy appearance, myofibre disarray, partial cross striation, cardiomyocytes hydropic degeneration together with vacuolation and nuclear alterations. Moreover, acute exposure to TiO₂ NPs induced the following testicular alterations: spermatocytes degeneration, spermatids sloughing and interstitial edema. The presented cardiac and testicular alterations were dose dependent. From the findings of the present study, it might be concluded that TiO₂ nanomaterials are capable of inducing acute cardiac and testicular damage that is dose dependent and could adversely affect the function of the vital organs.     

Electrospun nylon-6 spider-net like nanofiber mat containing TiO2 nanoparticles: A multifunctional nanocomposite textile material

In this study, electrospun nylon-6 spider-net like nanofiber mats containing TiO₂ nanoparticles (TiO₂ NPs) were successfully prepared. The nanofiber mats containing TiO₂ NPs were characterized by SEM, FE-SEM, TEM, XRD, TGA and EDX analyses. The results revealed that fibers in two distinct sizes (nano and subnano scale) were obtained with the addition of a small amount of TiO₂ NPs. In low TiO₂ content nanocomposite mats, these nanofiber weaves were found uniformly loaded with TiO₂ NPs on their wall. The presence of a small amount of TiO₂ NPs in nylon-6 solution was found to improve the hydrophilicity (antifouling effect), mechanical strength, antimicrobial and UV protecting ability of electrospun mats. The resultant nylon-6/TiO₂ antimicrobial spider-net like composite mat with antifouling effect may be a potential candidate for future water filter applications, and its improved mechanical strength and UV blocking ability will also make it a potential candidate for protective clothing.     

Mechanistic Investigation of the Biological Effects of SiO2, TiO2, and ZnO Nanoparticles on Intestinal Cells

Silicon dioxide (SiO₂), titanium dioxide (TiO₂), and zinc oxide (ZnO) are currently among the most widely used nanoparticles (NPs) in the food industry. This could potentially lead to unintended exposure of the gastrointestinal tract to these NPs. This study aims to investigate the potential side‐effects of these food‐borne NPs on intestinal cells and to mechanistically understand the observed biological responses. Among the panel of tested NPs, ZnO NPs are the most toxic. Consistently in all three tested intestinal cell models, ZnO NPs invoke the most inflammatory responses from the cells and induce the highest intracellular production of reactive oxygen species (ROS). The elevated ROS levels induce significant damage to the DNA of the cells, resulting in cell‐cycle arrest and subsequently cell death. In contrast, both SiO₂ and TiO₂ NPs elicit minimum biological responses from the intestinal cells. Overall, the study showcases the varying capability of the food‐borne NPs to induce a cellular response in the intestinal cells. In addition to physicochemical differences in the NPs, the genetic landscape of the intestinal cell models governs the toxicology profile of these food‐borne NPs.     

Hepatic Differentiation of Human Embryonic Stem Cells as Microscaled Multilayered Colonies Leading to Enhanced Homogeneity and Maturation

Directed differentiation of human embryonic stem cells (hESCs) towards hepatocyte‐like cells on planar tissue culture plates has been extensively investigated with great promise to provide alternative cell sources for drug metabolism/toxicity testing. Recently, hepatic differentiation of hESCs in 3D configuration with better mimicry of embryonic liver development represents incremental efforts to improve the differentiation efficiency and cellular maturation. However, most of the present 3D differentiation configurations involved interruptive operations during the multi‐staged differentiation process, which might impose unwanted influence on cellular differentiation. Most of the current researches resulted in generation of hepatocytes with high expression of AFP, which is minimally expressed in primary hepatocytes. Here, off‐the‐shelf micro‐stencil arrays are developed to generate adherent multilayered colonies composed of hESCs‐derived cells. Uninterrupted cellular differentiation and proliferation is achieved to recapitulate the continuous and multi‐stage liver development. Compared with conventional 2D format, the micro‐scaled multilayered colonies with uniform and defined sizes constrained within the microwells are composed of more homogenous and mature hepatocyte‐like cells with significantly lowered AFP expression and elevated hepatic functions. The multilayered colonies as novel 3D configuration for hepatic differentiation of hESCs represent a significant step toward efficient generation of functional hepatocytes for regenerative medicine and drug discovery.     

Analysis of the cytotoxicity of differentially sized titanium dioxide nanoparticles in murine MC3T3-E1 preosteoblasts

There is an increased use of nanophase titanium dioxide (TiO₂) in bone implants and scaffolds. However, nano-debris is generated at the bone-biomaterial interface. Therefore, TiO₂ nanoparticles (NPs) of many sizes were investigated for cytotoxic effects on murine MC3T3-E1 preosteoblasts. These TiO₂ NPs induced a time- and dose-dependent decrease in cell viability. There was a significant increase in lactate dehydrogenase (LDH) release, apoptosis and mitochondrial membrane permeability following short-term exposure of the cells to TiO₂ NPs. These NPs also increased granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) gene expression. Compared with the 32 nm TiO₂ NPs, 5 nm TiO₂ NPs were more toxic, induced more apoptosis, increased mitochondrial membrane permeability and stimulated more GM-CSF expression at a high concentration (≥100 μg/ml). The results implied that the differential toxicity was associated with variations in size, so more attention should be given to the toxicity of small NPs for the design of future materials for implantation.     

Resistive Switching of Individual,Chemically Synthesized TiO2 Nanoparticles

Resistively switching devices are considered promising for next‐generation nonvolatile random‐access memories. Today, such memories are fabricated by means of “top–down approaches” applying thin films sandwiched between nanoscaled electrodes. In contrast, this work presents a “bottom–up approach” disclosing for the first time the resistive switching (RS) of individual TiO₂ nanoparticles (NPs). The NPs, which have sizes of 80 and 350 nm, respectively, are obtained by wet chemical synthesis and thermally treated under oxidizing or vacuum conditions for crystallization, respectively. These NPs are deposited on a Pt/Ir bottom electrode and individual NPs are electrically characterized by means of a nanomanipulator system in situ, in a scanning electron microscope. While amorphous NPs and calcined NPs reveal no switching hysteresis, a very interesting behavior is found for the vacuum‐annealed, crystalline TiO_2–x NPs. These NPs reveal forming‐free RS behavior, dominantly complementary switching (CS) and, to a small degree, bipolar switching (BS) characteristics. In contrast, similarly vacuum‐annealed TiO₂ thin films grown by atomic layer deposition show standard BS behavior under the same conditions. The interesting CS behavior of the TiO_2–x NPs is attributed to the formation of a core–shell‐like structure by re‐oxidation of the reduced NPs as a unique feature.     

TiO2‐Decorated Graphite Nanoplatelet Nanocomposites for High‐Temperature Sensor Applications

Temperature and/or composition mapping inside high temperature energy conversion and storage devices are challenging, yet of critical importance to improve the material design for optimum performance. Here, the great potential of TiO₂ nanoparticle (NP)‐decorated graphite nanoplatelet (GNP) nanocomposites as high temperature thermal senors or gas sensors is reported. Effects of the GNP substrate on phonon confinement in Raman spectrum, grain growth, and phase stability of anatase TiO₂ NPs at high temperatures are systematically studied. Thermally sensitive Raman signatures, indicating the ultrafast grain growth of TiO₂ NPs in response to short thermal shock treatments (0.1–25 s) at high temperatures, are exploited for high temperature thermal sensing applications. A very high accuracy of nearly 98% in temperature measurements is demonstrated for a given short‐time thermal exposure. Thermal stability of anatase TiO₂ NPs against transformation into the rutile phase in TiO₂‐GNP nancomposites is substantially increased by controlling the surface area of the substrate, which would significantly improve the performance of TiO₂‐based high temperature gas sensors.     

Foodborne Titanium Dioxide Nanoparticles Induce Stronger Adverse Effects in Obese Mice than Non‐Obese Mice: Gut Microbiota Dysbiosis,Colonic Inflammation,and Proteome Alterations

The recent ban of titanium dioxide (TiO₂) as a food additive (E171) in France intensified the controversy on safety of foodborne‐TiO₂ nanoparticles (NPs). This study determines the biological effects of TiO₂ NPs and TiO₂ (E171) in obese and non‐obese mice. Oral consumption (0.1 wt% in diet for 8 weeks) of TiO₂ (E171, 112 nm) and TiO₂ NPs (33 nm) does not cause severe toxicity in mice, but significantly alters composition of gut microbiota, for example, increased abundance of Firmicutes phylum and decreased abundance of Bacteroidetes phylum and Bifidobacterium and Lactobacillus genera, which are accompanied by decreased cecal levels of short‐chain fatty acids. Both TiO₂ (E171) and TiO₂ NPs increase abundance of pro‐inflammatory immune cells and cytokines in the colonic mucosa, indicating an inflammatory state. Importantly, TiO₂ NPs cause stronger colonic inflammation than TiO₂ (E171), and obese mice are more susceptible to the effects. A microbiota transplant study demonstrates that altered fecal microbiota by TiO₂ NPs directly mediate inflammatory responses in the mouse colon. Furthermore, proteomic analysis shows that TiO₂ NPs cause more alterations in multiple pathways in the liver and colon of obese mice than non‐obese mice. This study provides important information on the health effects of foodborne inorganic nanoparticles.     

Diverse biotechnological applications of multifunctional titanium dioxide nanoparticles: An up‐to‐date review

Titanium dioxide (TiO₂) nanoparticles (NPs) are one of the topmost widely used metallic oxide nanoparticles. Whether present in naked form or doped with metals or polymers, TiO₂ NPs perform immensely important functions. However, the alteration in size and shape by doping results in improving the physical, chemical, and biological behaviour of TiO₂ NPs. Hence, the differential effects of various TiO₂ nanostructures including nanoflakes, nanoflowers, and nanotubes in various domains of biotechnology have been elucidated by researchers. Recently, the exponential growth of research activities regarding TiO₂ NPs has been observed owing to their chemical stability, low toxicity, and multifaceted properties. Because of their enormous abundance, plants, humans, and environment are inevitably exposed to TiO₂ NPs. These NPs play a significant role in improving agricultural attributes, removing environmental pollution, and upgrading the domain of nanomedicine. Therefore, the currently ongoing studies about the employment of TiO₂ NPs in enhancement of different aspects of agriculture, environment, and medicine have been extensively discussed in this review.     

Growth of ZnO nanorods on TiO2 nanoparticles films and their application to the electrode of dye-sensitized solar cells

A ZnO nanorods (NRs)/TiO₂ nanoparticles (NPs) film has been prepared by electrochemical deposition of ZnO NRs growth on P25 TiO₂ NPs film surfaces. It was found that ZnO NRs/TiO₂ NPs could significantly improve the efficiency of dye-sensitized solar cells owing to its relatively enhanced light-scattering capability and efficient charge transport efficiency. The overall energy-conversion efficiency (η) of 3.48 % was achieved by the formation of ZnO NRs/TiO₂ NPs film, which is 33 % higher than that formed by TiO₂ NPs alone (η = 2.62 %). The charge recombination behavior of cells was investigated by electrochemical impedance spectra, and the results showed that ZnO NRs/TiO₂ NPs film has the longer electron lifetime than TiO₂ NPs alone, which could facilitate the reduction of recombination processes and thus would promote the photocatalysis and solar cell performance.     

Inhibitory effect of TiO2 NPs on symbiotic arbuscular mycorrhizal fungi in plant roots

While nanoparticles (NPs) are known to exhibit antimicrobial properties, their effects on symbiotic arbuscular mycorrhizal fungi (AMF) in plant roots has to be carefully examined as NPs particularly of titanium dioxide (TiO₂) reach plant roots through varied sources such as fertilisers, plant protection products and other nanoproducts. The objective of the present study is to assess the effect of TiO₂ NPs on the symbiotic behaviour of AMF colonising rice (Oryza sativa L.) plants. Using sol–gel method, TiO₂ NPs with three different sizes were successfully synthesised employing doping. Characterisation of the prepared material was done by X‐ray powder diffraction and scanning electron microscopy. The synthesised materials were applied at 0, 25, 50 and 100 mg plant–1 to the rhizosphere of mycorrhizal rice plants maintained in pots. The study revealed that the prepared NPs had an inhibitory effect on arbuscular mycorrhizal symbiosis in plant roots. Development of AMF structures such as vesicles and arbuscules was significantly reduced in TiO₂ ‐doped NPs with a relatively more inhibition in 2% TiO₂ ‐doped NPs. Among the concentrations of TiO₂ NPs applied to different treatments, %F was significantly (P < 0.001) affected at medium to higher levels of application.Inspec keywords: nanoparticles, titanium compounds, antibacterial activity, sol‐gel processing, X‐ray diffraction, scanning electron microscopy, microorganisms, cellular biophysics, nanomedicineOther keywords: symbiotic arbuscular mycorrhizal fungi, plant roots, nanoparticles, antimicrobial properties, fertilisers, plant protection, nanoproducts, AMF colonising rice, sol‐gel method, X‐ray powder diffraction, scanning electron microscopy, mycorrhizal rice plants, rhizosphere, arbuscular mycorrhizal symbiosis, soil biota, TiO₂      

3D culturing of human adipose-derived stem cells enhances their pluripotency and differentiation abilities

Human mesenchymal stem cells,such as human adipose-derived stem cells(hASCs),are typically cultured on a two-dimensional(2 D)monolayer material surface,on which 2 D culturing methods are easily performed and time-saving.However,hASCs usually suffer from decreased pluripotency and differentiation ability when cultured with a 2 D monolayer culturing method compared to hASCs cultured with a three-dimensional(3 D)culturing method,such as suspension culture.In this study,we evaluated whether the pluripotency and differentiation ability of hASCs can be reversibly changed during sequential cultivation with 2 D and 3 D culturing processes.The hASCs cultivated with a 3 D culturing process after 2 D culture showed at least 2-fold enhanced pluripotency(Sox2,Nanog,and OCT4)compared with that of hASCs cultured with the 2 D culture process alone.Furthermore,hASCs obtained from the 3 D culture process expressed increased levels of differentiation markers of chondrocytes and osteoblasts compared with hASCs obtained from the 2 D culture process when hASCs were induced to differentiate.However,their pluripotency and differentiation ability were extensively reduced when hASCs were shifted from 3 D culture to 2 D culture and vice versa,which indicates that hASCs show reversibility in terms of their pluripotency and differentiation ability depending on their environment in 2 D and 3 D culture.The reversibility of pluripotency and differentiation ability were found to last for at least 5 passages in culture during the alternative and sequential culture of cells with 2 D and 3 D culturing processes.Our study revealed the importance of the culture microenvironment in maintaining the pluripotency and differentiation ability of hASCs,which may reduce the effects of the aging process in hASCs.We discuss whether the environment of stem cell culture(i.e.,2 D or 3 D cultivation)can affect stem cell fate in terms of pluripotency and differentiation reversibility.