Functional Plasmon-Activated Water Increases Akkermansia muciniphila Abundance in Gut Microbiota to Ameliorate Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is associated with dysbiosis and intestinal barrier dysfunction, as indicated by epithelial hyperpermeability and high levels of mucosal-associated bacteria. Changes in gut microbiota may be correlated with IBD pathogenesis. Additionally, microbe-based treatments could mitigate clinical IBD symptoms. Plasmon-activated water (PAW) is known to have an anti-inflammatory potential. In this work, we studied the association between the anti-inflammatory ability of PAW and intestinal microbes, thereby improving IBD treatment. We examined the PAW-induced changes in the colonic immune activity and microbiota of mice by immunohistochemistry and next generation sequencing, determined whether drinking PAW can mitigate IBD induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dysbiosis through mice animal models. The effects of specific probiotic species on mice with TNBS-induced IBD were also investigated. Experimental results indicated that PAW could change the local inflammation in the intestinal microenvironment. Moreover, the abundance of Akkermansia spp. was degraded in the TNBS-treated mice but elevated in the PAW-drinking mice. Daily rectal injection of Akkermansia muciniphila, a potential probiotic species in Akkermansia spp., also improved the health of the mice. Correspondingly, both PAW consumption and increasing the intestinal abundance of Akkermansia muciniphila can mitigate IBD in mice. These findings indicate that increasing the abundance of Akkermansia muciniphila in the gut through PAW consumption or other methods may mitigate IBD in mice with clinically significant IBD.

Modulation effect of tea consumption on gut microbiota.

Tea is one of the most popular beverages in the world and possesses a wide range of beneficial effects for human health. The modulation of tea on gut microbiota has gained much interest in recent years. The present study discussed the modulation effect of various types of tea on gut microbiota, which plays crucial roles in human health, as investigated by in vitro animal and human studies. The currently available findings from a total of 23 studies support the modulation effects of tea liquid, tea extract, and its major active components, including polyphenols, polysaccharides, and teasaponin, on gut microbiota. Overall, tea possesses prebiotic-like effect and can alleviate the gut microbiota dysbiosis induced by high-fat diet in gut microbiota, despite the detailed bacterial taxa may alter depending on the types of tea supplemented. Current evidence implies that the modulation effect on gut microbiota may be an important action mechanism underlying the beneficial effect of tea consumption in daily life and also the great potential of strategically chosen tea extract to develop functional foods.

Activating Hippo Pathway via Rassf1 by Ursolic Acid Suppresses the Tumorigenesis of Gastric Cancer.

The Hippo pathway is often dysregulated in many carcinomas, which results in various stages of tumor progression. Ursolic acid (UA), a natural compound that exists in many herbal plants, is known to obstruct cancer progression and exerts anti-carcinogenic effect on a number of human cancers. In this study, we aimed to examine the biological mechanisms of action of UA through the Hippo pathway in gastric cancer cells. MTT assay showed a decreased viability of gastric cancer cells after treatment with UA. Following treatment with UA, colony numbers and the sizes of gastric cancer cells were significantly diminished and apoptosis was observed in SNU484 and SNU638 cells. The invasion and migration rates of gastric cancer cells were suppressed by UA in a dose-dependent manner. To further determine the gene expression patterns that are related to the effects of UA, a microarray analysis was performed. Gene ontology analysis revealed that several genes, such as the Hippo pathway upstream target gene, ras association domain family (RASSF1), and its downstream target genes (MST1, MST2, and LATS1) were significantly upregulated by UA, while the expression of YAP1 gene, together with oncogenes (FOXM1, KRAS, and BATF), were significantly decreased. Similar to the gene expression profiling results, the protein levels of RASSF1, MST1, MST2, LATS1, and p-YAP were increased, whereas those of CTGF were decreased by UA in gastric cancer cells. The p-YAP expression induced in gastric cancer cells by UA was reversed with RASSF1 silencing. In addition, the protein levels in the Hippo pathway were increased in the UA-treated xenograft tumor tissues as compared with that in the control tumor tissues; thus, UA significantly inhibited the tumorigenesis of gastric cancer in vivo in xenograft animals. Collectively, UA diminishes the proliferation and metastasis of gastric cancer via the regulation of Hippo pathway through Rassf1, which suggests that UA can be used as a potential chemopreventive and therapeutic agent for gastric cancer.

Therapeutics for Inflammatory-Related Diseases Based on Plasmon-Activated Water: A Review.

It is recognized that the properties of liquid water can be markedly different from those of bulk one when it is in contact with hydrophobic surfaces or is confined in nano-environments. Because our knowledge regarding water structure on the molecular level of dynamic equilibrium within a picosecond time scale is far from completeness all of water's conventionally known properties are based on inert "bulk liquid water" with a tetrahedral hydrogen-bonded structure. Actually, the strength of water's hydrogen bonds (HBs) decides its properties and activities. In this review, an innovative idea on preparation of metastable plasmon-activated water (PAW) with intrinsically reduced HBs, by letting deionized (DI) water flow through gold-supported nanoparticles (AuNPs) under resonant illumination at room temperature, is reported. Compared to DI water, the created stable PAW can scavenge free hydroxyl and 2,2-diphenyl-1-picrylhydrazyl radicals and effectively reduce NO release from lipopolysaccharide-induced inflammatory cells. Moreover, PAW can dramatically induce a major antioxidative Nrf2 gene in human gingival fibroblasts. This further confirms its cellular antioxidative and anti-inflammatory properties. In addition, innovatively therapeutic strategy of daily drinking PAW on inflammatory-related diseases based on animal disease models is demonstrated, examples being chronic kidney disease (CKD), chronic sleep deprivation (CSD), and lung cancer.

Quantitative evaluation on activated property-tunable bulk liquid water with reduced hydrogen bonds using deconvoluted Raman spectroscopy.

Interesting properties of water with distinguishable hydrogen-bonding structure on interfacial phase or in confined environment have drawn wide attentions. However, these unique properties of water are only found within the interfacial phase and confined environment, thus, their applications are limited. In addition, quantitative evaluation on these unique properties associating with the enhancement of water's physical and chemical activities represents a notable challenge. Here we report a practicable production of free-standing liquid water at room temperature with weak hydrogen-bonded structure naming Au nanoparticles (NPs)-treated (AuNT) water via treating by plasmon-induced hot electron transfer occurred on resonantly illuminated gold NPs (AuNPs). Compared to well-known untreated bulk water (deionized water), the prepared AuNT water exhibits many distinct activities in generally physical and chemical reactions, such as high solubilities to NaCl and O2. Also, reducing interaction energy within water molecules provides lower overpotential and higher efficiency in electrolytic hydrogen production. In addition, these enhanced catalytic activities of AuNT water are tunable by mixing with deionized water. Also, most of these tunable activities are linearly proportional to its degree of nonhydrogen-bonded structure (DNHBS), which is derived from the O-H stretching in deconvoluted Raman spectrum.

Room-temperature sensor based on surface-enhanced Raman spectroscopy.

As reported in the literature, several factors, such as scattering cross sections, polarisability and wavelength suitability, contribute to increased SERS enhancement. In general, the advantage of surface-enhanced Raman scattering (SERS)-active Ag nanoparticles (NPs) is their higher SERS enhancement over Au NPs because the molar extinction coefficient of the Ag NPs is the highest of its kind among metals. Nevertheless, the corresponding SERS-active hot spots on Au are of inherently greater stability than on Ag. In this work, innovative temperature sensors based on SERS-active Au and Ag substrates prepared by sonoelectrochemical deposition-dissolution cycles (SEDDCs) are first reported. The SERS intensity of the model probe molecules of Rhodamine 6G (R6G) adsorbed on a SERS-active Ag substrate is monotonically increased from 25 to 50 °C. Moreover, this temperature-dependent intensity is linear with a slope of ca. 430 cps per °C between 25 to 45 °C. In addition, the reversibility and reusability of the developed temperature sensors are evaluated after the R6G-adsorbed sensors are alternately exposed to the temperatures of 25 and 45 °C in a sealed chamber. After every five cycles, the SERS spectra of treated substrates were recorded and compared with those of the as-prepared substrates. Experimental results indicate that SERS enhancement capability is mostly reversible based on 90% intensity of the Raman signal being maintained for the SERS-active Au substrate after 25 cycles (only 15 cycles for the Ag substrate).

Innovative fabrication of a Au nanoparticle-decorated SiO2 mask and its activity on surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) utilizing the well-defined localized surface plasmon resonance (LSPR) of Ag and Au nanoparticles (NPs) under resonant irradiation has emerged as a promising spectroscopy technique for providing vibrational information on trace molecules. The Raman scattering intensity from molecules close to the surface of these finely divided metals can be significantly enhanced by a factor of more than 10(6). In addition to the high sensitivity, the reproducibility of the SERS signal is also an important parameter for its reliable application. In this work, we report on the innovative and facile fabrication of a Au NP-decorated SiO2 mask coated on indium tin oxide (ITO) glass as a SERS array substrate. First, a highly ordered porous SiO2 mask with pore sizes of 350 nm in diameter and wall thickness of 60 nm was deposited on ITO glass by using spin coating. Then, Au NPs were controllably decorated into the pores of the conductive ITO glass-bottomed SiO2 mask by using sonoelectrochemical deposition-dissolution cycling (SEDDC). Experimental results indicate that the SERS effect of Rhodamine 6G (R6G) observed on this developed substrate increases with an increase in the deposition time of Au NPs in SEDDC. The corresponding optimal enhancement factor (EF) that is obtained is ca. 6.5 × 10(7). Significantly, this system achieves an optimal reproducibility under a medium-length deposition time of Au NPs in SEDDC with a relative standard deviation (RSD) of 12% for measurements of five spots on different areas. The low RSD of the SERS signal and the large EF suggest that the developed array system can serve as an excellent spectroscopy platform for practical applications in analytical chemistry.

Preparation of pure active water for auto-catalytic reactions performed in it.

In catalyzed electrochemical reactions, a general strategy is to modify electrode materials to increase the efficiency of the reaction. From the viewpoint of environmental protection, electrochemical reactions should be performed in an inert green water phase. In this study, we report active pure liquid water (named PV), which was collected from the condensed vapor of heated gold (Au)-containing plasmon-activated water (PAW) with a distinct structure of electron-doping and reduced hydrogen bonding (HB). The resulting PV also exhibited distinct properties of the formation of stronger intermolecular HB with alcohols, and notable activities in catalytic electrochemical reactions, compared to bulk deionized water (DIW). Moreover, the measured diffusion coefficients of water increased by ca. 30% in PV solutions. Two typical electrochemical reactions significantly increased peak currents observed in oxidation-reduction cycles (ORCs) with roughening of the Au substrate and in a model of reversible oxidation-reduction reactions on a platinum (Pt) substrate. Also, PV enhanced hydrogen evolution reactions (HERs) on catalytic Pt and inert stainless steel substrates in PV-based solutions at different pH values, compared to DIW. Moreover, these activities of PV were more marked, even better than those of PAW, when PV was collected under a higher heating rate used to heat PAW. Active pure PV has emerged as a promising green solvent applicable to various chemical reactions with more efficiency.

Combined Lycium barbarum Polysaccharides with Plasmon-Activated Water Affect IFN-γ/TNF-α Induced Inflammation in Caco-2 Cells.

The effects of Lycium barbarum polysaccharides (LBP) and plasmon-activated water (PAW) against IFN-γ/TNF-α induced inflammation in human colon Caco-2 cells were investigated. Cells were divided into the control, induction, LBP treatment (100-500 µg/mL), and combination groups with PAW. Inflammation was induced 24 h with 10 ng/mL IFN-γ when cell confluency reached >90%, and various doses of LBP with or without PAW were treated for 3 h, and subsequently 50 ng/mL TNF-α was added for another 24 h to provoke inflammation. Combination of LBP with PAW significantly decreased the secretion of IL-6 and IL-8. Cyclooxygenase-2 and inducible NO synthase expression was attenuated in all LBP-treated groups with or without PAW. NLRP3 inflammasome and related protein PYCARD expression were inhibited by LBP at the highest dose (500 µg/mL). All doses of LBP alone significantly decreased p-ERK expression, but combination with PAW increased p-ERK expression compared to those without PAW. Additionally, 250 and 500 µg/mL of LBP with or without PAW inhibited procaspase-3/caspase-3 expression. Therefore, LBP possesses anti-inflammation and anti-apoptosis by inhibiting the secretion of inflammatory cytokines and the expression of NLRP3 inflammasome-related protein. The combination with PAW exerts additive or synergistic effect on anti-inflammation.

Plasmon-activated water as a therapeutic strategy in Alzheimer's disease by altering gut microbiota.

Gut microbiota (GM) are involved in the pathophysiology of Alzheimer's disease (AD) and might correlate to the machinery of the gut-brain axis. Alteration of the GM profiles becomes a potential therapy strategy in AD. Here, we found that plasmon-activated water (PAW) therapy altered GM profile and reduced AD symptoms in APPswe/PS1dE9 transgenic mice (AD mice). GM profile showed the difference between AD and WT mice. PAW therapy in AD mice altered GM profile and fecal microbiota transplantation (FMT) reproduced GM profile in AD mice. PAW therapy and FMT in AD mice reduced cognitive decline and amyloid accumulation by novel object recognition (NOR) test and amyloid PET imaging. Immunofluorescent staining and western blot analysis of ß-amyloid (Aß) and phosphorylated (p)-tau in the brain of AD mice were reduced in PAW therapy and FMT. The inflammatory markers, interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α and pro-inflammatory indicator of arginase-1/CD86 ratio were also reduced. Furthermore, immunohistochemistry (IHC) analysis of occludin and claudin-5 in the intestine and AXL in the brain were increased to correlate with the abundant GM in PAW therapy and FMT. Our results showed the machinery of gut-brain axis, and PAW might be a potential therapeutic strategy in AD.

HCH6-1, an antagonist of formyl peptide receptor-1, exerts anti-neuroinflammatory and neuroprotective effects in cellular and animal models of Parkinson's disease.

Microglial activation-induced neuroinflammation contributes to onset and progression of sporadic and hereditary Parkinson's disease (PD). Activated microglia secrete pro-inflammatory and neurotoxic IL-1ß, IL-6 and TNF-α, which subsequently promote neurodegeneration. Formyl peptide receptor-1 (FPR1) of CNS microglia functions as pattern recognition receptor and is activated by N-formylated peptides, leading to microglial activation, induction of inflammatory responses and resulting neurotoxicity. In this study, it was hypothesized that FPR1 activation of microglia causes loss of dopaminergic neurons by activating inflammasome and upregulating IL-1ß, IL-6 or TNF-α and that FPR1 antagonist HCH6-1 exerts neuroprotective effect on dopaminergic neurons. FPR1 agonist fMLF induced activation of microglia cells by causing activation of NLRP3 inflammasome and upregulation and secretion of IL-1ß, IL-6 or TNF-α. Conditioned medium (CM) of fMLF-treated microglia cells, which contains neurotoxic IL-1ß, IL-6 and TNF-α, caused apoptotic death of differentiated SH-SY5Y dopaminergic neurons by inducing mitochondrial oxidative stress and activating pro-apoptotic signaling. FPR1 antagonist HCH6-1 prevented fMLF-induced activation of inflammasome and upregulation of pro-inflammatory cytokines in microglia cells. HCH6-1 co-treatment reversed CM of fMLF-treated microglia-induced apoptotic death of dopaminergic neurons. FPR1 antagonist HCH6-1 inhibited rotenone-induced upregulation of microglial marker Iba-1 protein level, cell death of dopaminergic neurons and motor impairment in zebrafish. HCH6-1 ameliorated rotenone-induced microglial activation, upregulation of FPR1 mRNA, activation of NLRP3 inflammasome, cell death of SN dopaminergic neurons and PD motor deficit in mice. Our results suggest that FPR1 antagonist HCH6-1 possesses anti-neuroinflammatory and neuroprotective effects on dopaminergic neurons by inhibiting microglial activation and upregulation of inflammasome activity and pro-inflammatory cytokines.

Surface-enhanced Raman scattering-active gold nanoparticles modified with a monolayer of silver film.

As shown in the literature, electrochemical underpotential deposition (UPD) offers the ability to deposit up to a monolayer of one metal onto a more noble metal with a flat surface. In this work, we develop an electrochemical pathway to prepare more surface-enhanced Raman scattering (SERS)-active substrates with Ag UPD-modified Au nanoparticles (NPs) by using sonoelectrochemical deposition-dissolution cycles (SEDDCs). Encouragingly, the SERS of Rhodamine 6G (R6G) adsorbed on these Ag UPD-modified Au NPs exhibits a higher intensity by ca. 12-fold magnitude, as compared with that of R6G adsorbed on unmodified Au NPs. The prepared SERS-active substrate demonstrates a large Raman scattering enhancement for R6G with a detection limit of 2 × 10(-14) M and an enhancement factor of 5.0 × 10(8). Also, the strategy proposed in this work to improve the SERS effects by using UPD Ag based on SEDDCs has an effect on the smaller probe molecules of 2,2'-bipyridine (BPy).

Improved stabilities on surface-enhanced Raman scattering-active Ag/Al2O3 films on substrates.

As is shown in the literature, surface-enhanced Raman scattering (SERS)-active Ag films obtained by the salting-out of Ag colloids from solutions with some salts, are popularly used to examine the structure of analytes. SERS-active Ag nanoparticles (NPs) demonstrate more significant SERS effects than Au NPs, however, problems regarding the stabilities of SERS-active Ag NPs remain to be overcome. In this work, Ag/Al(2)O(3) colloids were prepared in 0.1 M HNO(3) solutions containing Al(2)O(3) NPs with higher heat capacity by sonoelectrochemical methods. SERS-active Ag/Al(2)O(3) films deposited on glass slides were prepared by the addition of a saturated NaCl solution in the prepared Ag/Al(2)O(3) colloids-containing solution. In an acceptable sacrifice of Raman intensity by ca. 30% magnitude, the prepared Ag/Al(2)O(3) films markedly improved thermal stability by raising the operation temperature over 100 °C, compared to Ag films. Meanwhile, aging of SERS enhancement capability in an atmosphere of relative humidity (RH) of 50% and 20% (v/v) O(2) at 30 °C is significantly depressed using Ag/Al(2)O(3) films.

Size-controllable synthesis of surface-enhanced Raman scattering-active gold nanoparticles coated on TiO2.

As shown in the literature, gold nanoparticles (NPs) were popularly used in the fields of catalyst and surface-enhanced Raman scattering (SERS). In this work, size-controllable Au NPs coated on TiO(2) are synthesized by adjusting the pH of solutions based on sonoelectrochemical methods. The size-controlled Au NPs on TiO(2), ranging from 2 to 80 nm in diameter, can be obtained by varying the pH of solutions from 3 to 7 and placing the sample for 3 h before sonoelectrochemical reductions. The optimal particle sizes of Au NPs on TiO(2) to obtain the strongest SERS effects under an irradiation of 785 nm for probe molecules of adsorbed Rhodamine 6G (R6G) and deposited polypyrrole (PPy) are all ca. 60 nm.

Real-world treatment pattern and prognostic factors of stage IV lung squamous cell carcinoma patients.

Lung squamous cell carcinoma (LUSC) represents a minor proportion of nonsmall cell lung cancer (NSCLC) harboring a poor prognosis. Herein, retrospective medical record research was performed to investigate real-world treatment patterns and identify the prognostic factors among LUSC patients. A total of 173 patients with a median age of 68 years were enrolled for analysis. Males were predominant (n = 143, 83%) and current or ex-smokers contributed to 78% of the entire cohort. Pleura and lung were the most common metastatic sites, whereas brain metastasis was only 7%. After diagnosis, however, only 107 patients (62%) had received first-line chemotherapy. In the chemotherapy cohort, median progression-free survival (PFS) and overall survival (OS) were 3.9 and 11.1 months, respectively. After multivariable analysis, bone metastasis and the use of first-line single-agent chemotherapy independently predicted shorter PFS. For baseline characteristics, male sex, metastasis to lung, pleura, liver, and bone independently predicted worse OS. Regarding the treatment pattern, patients who had undergone standard first-line doublet therapy and employed targeted therapies after disease progression linked to longer OS. In the real world, even those who underwent chemotherapy still had poor outcome. The findings may help clinicians to orchestrate the treatment strategies for LUSC patients and provide further direction of large-scale studies.

Transferring Plasmon Effect on a Biological System: Expression of Biological Polymers in Chronic Rejection and Inflammatory Rat Model.

The plasmon-activated water (PAW) that reduces hydrogen bonds is made of deionized reverse osmosis water (ROW). However, compared with ROW, PAW has a significantly higher diffusion coefficient and electron transfer rate constant in electrochemical reactions. PAW has a boiling point of97 °C and specific heat of0.94; the energy of PAW is also 1121 J/mol higher than ordinary water. The greater the force of hydrogen bonds between H2O, the larger the volume of the H2O cluster, and the easier it is to lose the original characteristics. The hydrogen bonding force of PAW is weak, so the volume of its cluster is small, and it exists in a state very close to a single H2O. PAW has a high permeability and diffusion rate, which can improve the needs of biological applications and meet the dependence of biological organisms on H2O when performing physiological functions. PAW can successfully remove free radicals, and efficiently reduce lipopolysaccharide (LPS)-induced monocytes to release nitric oxide. PAW can induce expression of the antioxidant gene Nrf2 in human gingival fibroblasts, lower amyloid burden in mice with Alzheimer's disease, and decrease metastasis in mice grafted with Lewis lung carcinoma cells. Because the transferring plasmon effect may improve the abnormality of physiological activity in a biological system, we aimed to evaluate the influence of PAW on orthotopic allograft transplantation (OAT)-induced vasculopathy in this study. Here, we demonstrated that daily intake of PAW lowered the progression of vasculopathy in OAT-recipient ACI/NKyo rats by inhibiting collagen accumulation, proliferation of smooth muscle cells and fibroblasts, and T lymphocyte infiltration in the vessel wall. The results showed reduced T and B lymphocytes, plasma cells, and macrophage activation in the spleen of the OAT-recipient ACI/NKyo rats that were administered PAW. In contrast to the control group, the OAT-recipient ACI/NKyo rats that were administered PAW exhibited higher mobilization and levels of circulating endothelial progenitor cells associated with vessel repair. We use the transferring plasmon effect to adjust and maintain the biochemical properties of water, and to meet the biochemical demand of organisms. Therefore, this study highlights the therapeutic roles of PAW and provides more biomedical applicability for the transferring plasmon effect.

Functional variant rs17525453 within RAB35 gene promoter is possibly associated with increased risk of Parkinson's disease in Taiwanese population.

Our previous study suggests that upregulated RAB35 is implicated in etiology of Parkinson's disease (PD). We hypothesized that upregulated RAB35 results from single nucleotide polymorphisms (SNPs) in RAB35 gene promoter. We identified SNPs within RAB35 gene promoter by analyzing DNA samples of discovery cohort and validation cohort. SNP rs17525453 within RAB35 gene promoter (T>C at position of -66) was significantly associated with idiopathic PD patients. Compared to normal controls, sporadic PD patients had higher C allele frequency. CC and CT genotype significantly increased risk of PD compared with TT genotype. SNP rs17525453 within RAB35 gene promoter leads to formation of transcription factor TFII-I binding site. Results of EMSA and supershift assay indicated that TFII-I binds to rs17525453 sequence of RAB35 gene promoter. Luciferase reporter assays showed that rs17525453 variant of RAB35 gene promoter possesses an augmented transcriptional activity. Our results suggest that functional variant rs17525453 within RAB35 gene promoter is likely to enhance transcriptional activity and upregulate RAB35 protein, which could lead to increased risk of PD in Taiwanese population.

Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells.

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are 'true' totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely 'cluster 3', as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.