Chromatin-Remodeling Components of the BAF Complex Facilitate Reprogramming.

Reprogramming of somatic cells achieved by combination of the four transcription factors Oct4, Sox2, Klf4, and c-Myc has very low efficiency. To increase the reprogramming efficiency and better understand the process, we sought to identify factors that mediate reprogramming with higher efficiency. We established an assay to screen nuclear fractions from extracts of pluripotent mouse cells based on Oct4 reactivation. Using proteomics, we identified components of the ATP-dependent BAF chromatin-remodeling complex, which significantly increases reprogramming efficiency when used together with the four factors. The reprogrammed cells could transmit to the germline and exhibited pluripotency. Reprogramming remained highly efficient when c-Myc was not present but BAF components were overexpressed. BAF complex components mediate this effect by facilitating enhanced Oct4 binding to target promoters during reprogramming. Thus, somatic cell reprogramming using chromatin-remodeling molecules represents an efficient method of generating reprogrammed cells.

Epiblast stem cell subpopulations represent mouse embryos of distinct pregastrulation stages.

Embryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast.

3D hepatic organoid production from human pluripotent stem cells.

Hepatic organoids might provide a golden opportunity for realizing precision medicine in various hepatic diseases. Previously described hepatic organoid protocols from pluripotent stem cells rely on complicated multiple differentiation steps consisting of both 2D and 3D differentiation procedures. Therefore, the spontaneous formation of hepatic organoids from 2D monolayer culture is associated with a low-throughput production, which might hinder the standardization of hepatic organoid production and hamper the translation of this technology to the clinical or industrial setting. Here we describe the stepwise and fully 3D production of hepatic organoids from human pluripotent stem cells. We optimized every differentiation step by screening for optimal concentrations and timing of differentiation signals in each differentiation step. Hepatic organoids are stably expandable without losing their hepatic functionality. Moreover, upon treatment of drugs with known hepatotoxicity, we found hepatic organoids are more sensitive to drug-induced hepatotoxicity compared with 2D hepatocytes differentiated from PSCs, making them highly suitable for in vitro toxicity screening of drug candidates. The standardized fully 3D protocol described in the current study for producing functional hepatic organoids might serve as a novel platform for the industrial and clinical translation of hepatic organoid technology.

Thyroid-Friendly Soft Materials as 3D Cell Culture Tool for Stimulating Thyroid Cell Function.

The disruption of thyroid hormones because of chemical exposure is a significant societal problem. Chemical evaluations of environmental and human health risks are conventionally based on animal experiments. However, owing to recent breakthroughs in biotechnology, the potential toxicity of chemicals can now be evaluated using 3D cell cultures. In this study, the interactive effects of thyroid-friendly soft (TS) microspheres on thyroid cell aggregates are elucidated and their potential as a reliable toxicity assessment tool is evaluated. Using state-of-the-art characterization methods coupled with cell-based analysis and quadrupole time-of-flight mass spectrometry, it is shown that TS-microsphere-integrated thyroid cell aggregates exhibit improved thyroid function. Specifically, the responses of zebrafish embryos, which are used for thyroid toxicity analysis, and the TS-microsphere-integrated cell aggregates to methimazole (MMI), a known thyroid inhibitor, are compared. The results show that the thyroid hormone disruption response of the TS-microsphere-integrated thyroid cell aggregates to MMI is more sensitive compared with those of the zebrafish embryos and conventionally formed cell aggregates. This proof-of-concept approach can be used to control cellular function in the desired direction and hence evaluate thyroid function. Thus, the proposed TS-microsphere-integrated cell aggregates may yield new fundamental insights for advancing in vitro cell-based research.

Permissive epigenomes endow reprogramming competence to transcriptional regulators.

Identifying molecular and cellular processes that regulate reprogramming competence of transcription factors broadens our understanding of reprogramming mechanisms. In the present study, by a chemical screen targeting major epigenetic pathways in human reprogramming, we discovered that inhibiting specific epigenetic roadblocks including disruptor of telomeric silencing 1-like (DOT1L)-mediated H3K79/K27 methylation, but also other epigenetic pathways, catalyzed by lysine-specific histone demethylase 1A, DNA methyltransferases and histone deacetylases, allows induced pluripotent stem cell generation with almost all OCT factors. We found that simultaneous inhibition of these pathways not only dramatically enhances reprogramming competence of most OCT factors, but in fact enables dismantling of species-dependent reprogramming competence of OCT6, NR5A1, NR5A2, TET1 and GATA3. Harnessing these induced permissive epigenetic states, we performed an additional screen with 98 candidate genes. Thereby, we identified 25 transcriptional regulators (OTX2, SIX3, and so on) that can functionally replace OCT4 in inducing pluripotency. Our findings provide a conceptual framework for understanding how transcription factors elicit reprogramming in dependency of the donor cell epigenome that differs across species.

Non-coding de novo mutations in chromatin interactions are implicated in autism spectrum disorder.

Three-dimensional chromatin interactions regulate gene expressions. The significance of de novo mutations (DNMs) in chromatin interactions remains poorly understood for autism spectrum disorder (ASD). We generated 813 whole-genome sequences from 242 Korean simplex families to detect DNMs, and identified target genes which were putatively affected by non-coding DNMs in chromatin interactions. Non-coding DNMs in chromatin interactions were significantly involved in transcriptional dysregulations related to ASD risk. Correspondingly, target genes showed spatiotemporal expressions relevant to ASD in developing brains and enrichment in biological pathways implicated in ASD, such as histone modification. Regarding clinical features of ASD, non-coding DNMs in chromatin interactions particularly contributed to low intelligence quotient levels in ASD probands. We further validated our findings using two replication cohorts, Simons Simplex Collection (SSC) and MSSNG, and showed the consistent enrichment of non-coding DNM-disrupted chromatin interactions in ASD probands. Generating human induced pluripotent stem cells in two ASD families, we were able to demonstrate that non-coding DNMs in chromatin interactions alter the expression of target genes at the stage of early neural development. Taken together, our findings indicate that non-coding DNMs in ASD probands lead to early neurodevelopmental disruption implicated in ASD risk via chromatin interactions.

Recent Advances in Marine Biomaterials Tailored and Primed for the Treatment of Damaged Soft Tissues.

The inherent self-repair abilities of the body often fall short when it comes to addressing injuries in soft tissues like skin, nerves, and cartilage. Tissue engineering and regenerative medicine have concentrated their research efforts on creating natural biomaterials to overcome this intrinsic healing limitation. This comprehensive review delves into the advancement of such biomaterials using substances and components sourced from marine origins. These marine-derived materials offer a sustainable alternative to traditional mammal-derived sources, harnessing their advantageous biological traits including sustainability, scalability, reduced zoonotic disease risks, and fewer religious restrictions. The use of diverse engineering methodologies, ranging from nanoparticle engineering and decellularization to 3D bioprinting and electrospinning, has been employed to fabricate scaffolds based on marine biomaterials. Additionally, this review assesses the most promising aspects in this field while acknowledging existing constraints and outlining necessary future steps for advancement.

Improving the developmental competences of porcine parthenogenetic embryos by Notoginsenoside R1-induced enhancement of mitochondrial activity and alleviation of proapoptotic events.

Notoginsenoside R1 (NGR1), derived from the Panax notoginseng root and rhizome, exhibits diverse pharmacological influences on the brain, neurons, and osteoblasts, such as antioxidant effects, mitochondrial function protection, energy metabolism regulation, and inhibition of oxygen radicals, apoptosis, and cellular autophagy. However, its effect on early porcine embryonic development remains unclear. Therefore, we investigated NGR1's effects on blastocyst quality, reactive oxygen species (ROS) levels, glutathione (GSH) levels, mitochondrial function, and embryonic development-related gene expression in porcine embryos by introducing NGR1 during the in vitro culture (IVC) of early porcine embryos. Our results indicate that an addition of 1 µM NGR1 significantly increased glutathione (GSH) levels, blastocyst formation rate, and total cell number and proliferation capacity; decreased ROS levels and apoptosis rates in orphan-activated porcine embryos; and improved intracellular mitochondrial distribution, enhanced membrane potential, and reduced autophagy. In addition, pluripotency-related factor levels were elevated (NANOG and octamer-binding transcription factor 4 [OCT4]), antioxidant-related genes were upregulated (nuclear factor-erythroid 2-related factor 2 [NRF2]), and apoptosis- (caspase 3 [CAS3]) and autophagy-related genes (light chain 3 [LC3B]) were downregulated. These results indicate that NGR1 can enhance early porcine embryonic development by protecting mitochondrial function.

Principles and Biomedical Application of Graphene Family Nanomaterials.

Two-dimensional graphene family nanomaterials (GFNs) are extensively studied by the researchers for their quantum size effect, large surface area, numerous reactive functional sites, and biocompatibility. The hybrid materials of GFNs exhibit an increased level of mechanical strength, optical, electronic, and catalytic activity due to their incorporation. The application of GFNs in the energy, environment, electric and electronic, personal care, and health sectors is abundant, which is not only by their unique physicochemical properties but also by their ease and large production by various synthetic approaches and economically inexpensiveness. Their general biomedical applications include bioimaging, biosensing, drug delivery, tissue engineering, killing the microbes, and demolishing the cancer tumor. The first chapter of this book describes definitions, synthetic methods, unique properties, and biomedical applications of GFNs, including graphene, graphene oxide, and reduced graphene oxide.

Reflections and Outlook on Multifaceted Biomedical Applications of Graphene.

Two-dimensional nanomaterials have been widely explored by researchers due to their nanosized thickness and quantum size effect. They were layered double hydroxides, transition metal dichalcogenides, transition metal oxides, and synthetic silicate clays. Among the 2D nanomaterials, graphene and their derivatives were investigated extensively at first as they exhibited exceptional conductivity and a zero-band gap semimetal nature. Though graphene family nanomaterials (GFNs) were utilized for several physicochemical applications, including electronic, electric, mechanic, photonic, magnetic, and catalytic devices, their biomedical applications are still meritorious. Biosensor, bioimaging, drug delivery, tumor ablation, and tissue regeneration are some of them. The outlook of the present book chapters encompasses the preparation of GFNs, physicochemical properties, biomedical applications, biosafety, and their future directions.

Graphene-Based Nanomaterials for Biomedical Imaging.

Graphene is sp2-hybridized carbon structure-based two-dimensional (2D) sheet. Graphene-based nanomaterials possess several features such as unique mechanical, electronic, thermal, and optical properties, high specific surface area, versatile surface functionalization, and biocompatibility, which attracted researcher's interests in various fields including biomedicine. In this chapter, we particularly focused on the biomedical imaging applications of graphene-based nanomaterials like graphene oxide (GO), reduced graphene oxide (rGO), graphene quantum dots (GQDs), graphene oxide quantum dots (GOQDs), and other derivatives, which utilize their outstanding optical properties. There are some biomedical imaging modalities using Graphene-based Nanomaterials, among which we will highlight fluorescence imaging, Raman imaging, magnetic resonance imaging, and photoacoustic imaging. We also discussed the brief perspectives and future application related to them.

Differential Toxicity of Graphene Family Nanomaterials Concerning Morphology.

Graphene family nanomaterials (GFNs) are well-known carbonaceous materials, which find application in several fields like optoelectronics, photocatalysis, nanomedicine, and tissue regeneration. Despite possessing many advantages in biomedical applications, GFNs exhibited toxicity depending on various parameters including dosage, size, exposure time, and kinds of administration. GFNS are majorly classified into nanosheets, quantum dots, nanoplatelets, and nanoribbons based on morphology. Understanding the toxic effects of GFNs would provide new suggestions as to how the materials can be utilized effectively. Hence, we are summarizing here some of the recent findings in cellular and animal level toxicity studies of GFNs on the perspective of their different morphologies. Notwithstanding, we highlight progress, challenges, and new toxicological approaches to ensure biosafety of GFNs for future directions.

Functional Graphene Nanomaterials-Based Hybrid Scaffolds for Osteogenesis and Chondrogenesis.

With the emerging trends and recent advances in nanotechnology, it has become increasingly possible to overcome current hurdles for bone and cartilage regeneration. Among the wide type of nanomaterials, graphene (G) and its derivatives (graphene-based materials, GBMs) have been highlighted due to the specific physicochemical and biological properties. In this review, we present the recent development of GBM-based scaffolds for bone and cartilage engineering, focusing on the formulation/shape/size-dependent characteristics, types of scaffold and modification, biocompatibility, bioactivity and underlying mechanism, drawback and prospect of each study. From the findings described herein, mechanical property, biocompatibility, osteogenic and chondrogenic property of GBM-based scaffolds could be significantly enhanced through various scaffold fabrication methods and conjugation with polymers/nanomaterials/drugs. In conclusion, the results presented in this review support the promising prospect of using GBM-based scaffolds for improved bone and cartilage tissue engineering. Although GBM-based scaffolds have some limitations to be overcome by future research, we expect further developments to provide innovative results and improve their clinical potential for bone and cartilage regeneration.

Graphene: A Promising Theranostic Agent.

Graphene has drawn tremendous interest in the field of nanoscience as a superior theranostic agent owing to its high photostability, aqueous solubility, and low toxicity. This monoatomic thick building block of a carbon allotrope exhibits zero to two-dimensional characteristics with a unique size range within the nanoscale. Their high biocompatibility, quantum yield, and photoluminescent properties make them more demandable in biomedical research. Its application in biomedical sciences has been limited due to its small-scale production. Large-scale production with an easy synthesis process is urgently required to overcome the problem associated with its translational application. Despite all possible drawbacks, the graphene-based drug/gene delivery system is gaining popularity day by day. To date, various studies suggested its application as a theranostic agent for target-specific delivery of chemotherapeutics or antibiotics against various diseases like cancer, Alzheimer's diseases, multidrug resistance diseases, and more. Also, studying the toxicological profile of graphene derivatives is very important before starting its practical use in clinical applications. This chapter has tried to abbreviate several methods and their possible incoming perspective as claimed by researchers for mass production and amplifying graphene-based treatment approaches.

Role of Graphene Family Nanomaterials in Skin Wound Healing and Regeneration.

Owing to astonishing properties such as the large surface area to volume ratio, mechanical stability, antimicrobial property, and collagen crosslinking, graphene family nanomaterials (GFNs) have been widely used in various biomedical applications including tissue regeneration. Many review literatures are available to compile the role of GFNs in cardiac, bone, and neuronal tissue regeneration. However, the contribution of GFNs in skin wound healing and tissue regeneration was not yet discussed. In the present review, we have highlighted the properties of GFNs and their application in skin wound healing. In addition, we have included challenges and future directions of GFNs in skin tissue regeneration in the portion of conclusion and perspectives.

Dynamic Change of R-Loop Implicates in the Regulation of Zygotic Genome Activation in Mouse.

In mice, zygotic genome activation (ZGA) occurs in two steps: minor ZGA at the one-cell stage and major ZGA at the two-cell stage. Regarding the regulation of gene transcription, minor ZGA is known to have unique features, including a transcriptionally permissive state of chromatin and insufficient splicing processes. The molecular characteristics may originate from extremely open chromatin states in the one-cell stage zygotes, yet the precise underlying mechanism has not been well studied. Recently, the R-loop, a triple-stranded nucleic acid structure of the DNA/RNA hybrid, has been implicated in gene transcription and DNA replication. Therefore, in the present study, we examined the changes in R-loop dynamics during mouse zygotic development, and its roles in zygotic transcription or DNA replication. Our analysis revealed that R-loops persist in the genome of metaphase II oocytes and preimplantation embryos from the zygote to the blastocyst stage. In particular, zygotic R-loop levels dynamically change as development proceeds, showing that R-loop levels decrease as pronucleus maturation occurs. Mechanistically, R-loop dynamics are likely linked to ZGA, as inhibition of either DNA replication or transcription at the time of minor ZGA decreases R-loop levels in the pronuclei of zygotes. However, the induction of DNA damage by treatment with anticancer agents, including cisplatin or doxorubicin, does not elicit genome-wide changes in zygotic R-loop levels. Therefore, our study suggests that R-loop formation is mechanistically associated with the regulation of mouse ZGA, especially minor ZGA, by modulating gene transcription and DNA replication.

INO80 Is Required for the Cell Cycle Control, Survival, and Differentiation of Mouse ESCs by Transcriptional Regulation.

Precise regulation of the cell cycle of embryonic stem cells (ESCs) is critical for their self-maintenance and differentiation. The cell cycle of ESCs differs from that of somatic cells and is different depending on the cell culture conditions. However, the cell cycle regulation in ESCs via epigenetic mechanisms remains unclear. Here, we showed that the ATP-dependent chromatin remodeler Ino80 regulates the cell cycle genes in ESCs under primed conditions. Ino80 loss led to a significantly extended length of the G1-phase in ESCs grown under primed culture conditions. Ino80 directly bound to the transcription start site and regulated the expression of cell cycle-related genes. Furthermore, Ino80 loss induced cell apoptosis. However, the regulatory mechanism of Ino80 in differentiating ESC cycle slightly differed; an extended S-phase was detected in differentiating inducible Ino80 knockout ESCs. RNA-seq analysis of differentiating ESCs revealed that the expression of genes associated with organ development cell cycle is persistently altered in Ino80 knockout cells, suggesting that cell cycle regulation by Ino80 is not limited to undifferentiated ESCs. Therefore, our study establishes the function of Ino80 in ESC cycle via transcriptional regulation, at least partly. Moreover, this Ino80 function may be universal to other cell types.

Generation of homogeneous midbrain organoids with in vivo-like cellular composition facilitates neurotoxin-based Parkinson's disease modeling.

Recent studies have demonstrated the generation of midbrain-like organoids (MOs) from human pluripotent stem cells. However, the low efficiency of MO generation and the relatively immature and heterogeneous structures of the MOs hinder the translation of these organoids from the bench to the clinic. Here we describe the robust generation of MOs with homogeneous distribution of midbrain dopaminergic (mDA) neurons. Our MOs contain not only mDA neurons but also other neuronal subtypes as well as functional glial cells, including astrocytes and oligodendrocytes. Furthermore, our MOs exhibit mDA neuron-specific cell death upon treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, indicating that MOs could be a proper human model system for studying the in vivo pathology of Parkinson's disease (PD). Our optimized conditions for producing homogeneous and mature MOs might provide an advanced patient-specific platform for in vitro disease modeling as well as for drug screening for PD.

Development of Two-Dimensional Nanomaterials Based Electrochemical Biosensors on Enhancing the Analysis of Food Toxicants.

In recent times, food safety has become a topic of debate as the foodborne diseases triggered by chemical and biological contaminants affect human health and the food industry's profits. Though conventional analytical instrumentation-based food sensors are available, the consumers did not appreciate them because of the drawbacks of complexity, greater number of analysis steps, expensive enzymes, and lack of portability. Hence, designing easy-to-use tests for the rapid analysis of food contaminants has become essential in the food industry. Under this context, electrochemical biosensors have received attention among researchers as they bear the advantages of operational simplicity, portability, stability, easy miniaturization, and low cost. Two-dimensional (2D) nanomaterials have a larger surface area to volume compared to other dimensional nanomaterials. Hence, researchers nowadays are inclined to develop 2D nanomaterials-based electrochemical biosensors to significantly improve the sensor's sensitivity, selectivity, and reproducibility while measuring the food toxicants. In the present review, we compile the contribution of 2D nanomaterials in electrochemical biosensors to test the food toxicants and discuss the future directions in the field. Further, we describe the types of food toxicity, methodologies quantifying food analytes, how the electrochemical food sensor works, and the general biomedical properties of 2D nanomaterials.

Carbon Dots-Mediated Fluorescent Scaffolds: Recent Trends in Image-Guided Tissue Engineering Applications.

Regeneration of damaged tissues or organs is one of the significant challenges in tissue engineering and regenerative medicine. Many researchers have fabricated various scaffolds to accelerate the tissue regeneration process. However, most of the scaffolds are limited in clinical trials due to scaffold inconsistency, non-biodegradability, and lack of non-invasive techniques to monitor tissue regeneration after implantation. Recently, carbon dots (CDs) mediated fluorescent scaffolds are widely explored for the application of image-guided tissue engineering due to their controlled architecture, light-emitting ability, higher chemical and photostability, excellent biocompatibility, and biodegradability. In this review, we provide an overview of the recent advancement of CDs in terms of their different synthesis methods, tunable physicochemical, mechanical, and optical properties, and their application in tissue engineering. Finally, this review concludes the further research directions that can be explored to apply CDs in tissue engineering.