The Construction of a Multi-Gene Risk Model for Colon Cancer Prognosis and Drug Treatments Prediction.

In clinical practice, colon cancer is a prevalent malignant tumor of the digestive system, characterized by a complex and progressive process involving multiple genes and molecular pathways. Historically, research efforts have primarily focused on investigating individual genes; however, our current study aims to explore the collective impact of multiple genes on colon cancer and to identify potential therapeutic targets associated with these genes. For this research, we acquired the gene expression profiles and RNA sequencing data of colon cancer from TCGA. Subsequently, we conducted differential gene expression analysis using R, followed by GO and KEGG pathway enrichment analyses. To construct a protein-protein interaction (PPI) network, we selected survival-related genes using the log-rank test and single-factor Cox regression analysis. Additionally, we performed LASSO regression analysis, immune infiltration analysis, mutation analysis, and cMAP analysis, as well as an investigation into ferroptosis. Our differential expression and survival analyses identified 47 hub genes, and subsequent LASSO regression analysis refined the focus to 23 key genes. These genes are closely linked to cancer metastasis, proliferation, apoptosis, cell cycle regulation, signal transduction, cancer microenvironment, immunotherapy, and neurodevelopment. Overall, the hub genes discovered in our study are pivotal in colon cancer and are anticipated to serve as important biological markers for the diagnosis and treatment of the disease.

Recent advances in understanding the regulatory mechanism of plasma membrane H+-ATPase through the brassinosteroid signaling pathway.

The polyhydroxylated steroid phytohormone brassinosteroids (BRs) control many aspects of plant growth, development and responses to environmental changes. Plasma membrane (PM) H+-ATPase, the well-known PM proton pump, is a central regulator in plant physiology, which mediates not only plant growth and development, but also adaptation to stresses. Recent studies highlight that PM H+-ATPase is at least partly regulated via the BR signaling. Firstly, the BR cell surface receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and multiple key components of BR signaling directly or indirectly influence PM H+-ATPase activity. Secondly, the SMALL AUXIN UP RNA (SAUR) gene family physically interacts with BRI1 to enhance organ development of Arabidopsis by activating PM H+-ATPase. Thirdly, RNA-sequencing (RNA-seq) assays showed that the expression of some SAUR genes is upregulated under the light or sucrose conditions, which is related to the phosphorylation state of the penultimate residue of PM H+-ATPase in a time-course manner. In this review, we describe the structural and functional features of PM H+-ATPase, and summarize recent progress toward understanding the regulatory mechanism of PM H+-ATPase by BRs, and briefly introduce how PM H+-ATPase activity is modulated by its own biterminal regions and the post-translational modifications.

Profiling DNA Cargos in Single Extracellular Vesicles via Hydrogel-Based Droplet Digital Multiple Displacement Amplification.

Due to the substantial heterogeneity among extracellular vesicle (EV) subpopulations, single-EV analysis has the potential to elucidate the mechanisms behind EV biogenesis and shed light on the myriad functions, leading to the development of novel diagnostics and therapeutics. While many studies have been devoted to reveal between-EV variations in surface proteins and RNAs, DNA cargos (EV-DNA) have received little attention. Here, we report a hydrogel-based droplet digital multiple displacement amplification approach for the comprehensive analysis of EV-DNA at the single-EV level. Single EVs are dispersed in thousands of hydrogel droplets and lysed for DNA amplification and identification. The droplet microfluidics strategy empowers the assay with single-molecule sensitivity and capability for absolute quantification of DNA-containing EVs. In particular, our findings indicate that 5-40% EVs are associated with DNA, depending on the cell of origin. Large EVs exhibit a higher proportion of DNA-containing EVs and a more substantial presence of intraluminal DNA, compared to small EVs. These DNA-containing EVs carry multiple DNA fragments on average. Furthermore, both double-stranded DNA and single-stranded DNA were able to be detected at the single-EV level. Utilizing this method, the abundance, distribution, and biophysical properties of EV-DNA in various EV populations are evaluated. The DNA level within EVs provides insight into the status of the originating cells and offers valuable information on the outcomes of anticancer treatments. The utilization of single-EV analysis for EV-DNA holds significant promise for early cancer detection and treatment response monitoring.

PEX26 Functions as a Metastasis Suppressor in Colorectal Cancer.

BACKGROUND/AIMS: Aberrant Peroxisomal Biogenesis Factor 26 (PEX26) occurs in multiple cell process. However, the role of PEX26 in colorectal cancer (CRC) development remains unknown. We aimed to study PEX26 expression, regulation, and function in CRC cells. METHODS: Using the bioinformatic analysis, real-time quantitative PCR, and immunohistochemistry staining, we detected the expression of PEX26 in CRC and normal tissues. We performed functional experiments in vitro to elucidate the effect of PEX26 on CRC cells. We analyzed the RNA-seq data to reveal the downstream regulating network of PEX26. RESULTS: PEX26 is significantly down-regulated in CRC and its low expression correlates with the poor overall survival of CRC patients. We further demonstrated that PEX26 over-expression inhibits the ability of CRC cell migration, invasion, and epithelial-mesenchymal transition (EMT), while PEX26 knockdown promotes the malignant phenotypes of migration, invasion, and EMT via activating the Wnt pathway. CONCLUSION: Overall, our results showed that the loss of PEX26 contributes to the malignant phenotype of CRC. PEX26 may serve as a novel metastasis repressor for CRC.

Novel Long Non-Coding RNA (lncRNA) Transcript AL137782.1 Promotes the Migration of Normal Lung Epithelial Cells through Positively Regulating LMO7.

The role of long non-coding RNA (lncRNAs) in biological processes remains poorly understood, despite their significant impact. Our previous research discovered that the expression of AL137782.1, a long transcript of the novel lncRNA ENSG00000261553, is upregulated in lung epithelial cells upon exposure to microbes. Furthermore, the expression of AL137782.1 exhibits variability between para-cancerous and lung adenocarcinoma samples. These findings imply that this lncRNA may play a role in both normal lung epithelial cellular processes and pathophysiology. To elucidate the function of AL137782.1 in lung epithelial cells, we utilized bioinformatics retrieval and analysis to examine its expression. We then analyzed its subcellular localization using fluorescence in situ hybridization (FISH) and subcellular fractionation. Through rapid amplification of cDNA ends (RACE), we confirmed the presence of a 4401 nt lncRNA AL137782.1 in lung epithelial cells. Moreover, we discovered that this lncRNA positively regulates both mRNA and the protein expression of LMO7, a protein that may regulate the cell migration of normal lung epithelial cells. Although the overexpression of AL137782.1 has been shown to enhance the migration of both normal lung epithelial cells and lung adenocarcinoma cells in vitro, our study revealed that the expression of this lncRNA was significantly decreased in lung cancers compared to adjacent tissues. This suggests that the cell migration pattern regulated by the AL137782.1-LMO7 axis is more likely to occur in normal lung epithelial cells, rather than being a pathway that promotes lung cancer cell migration. Therefore, our study provides new insights into the mechanism underlying cell migration in human lung epithelial cells. This finding may offer a potential strategy to enhance normal lung epithelial cell migration after lung injury.

Extracellular Vesicles Released from Macrophages Infected with Mycoplasma pneumoniae Stimulate Proinflammatory Response via the TLR2-NF-κB/JNK Signaling Pathway.

Mycoplasma pneumoniae (M. pneumoniae, Mp) is an intracellular pathogen that causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans and can infect and survive in the host cells leading to excessive immune responses. Extracellular vesicles (EVs) from host cells carry components of pathogens to recipient cells and play a role in intercellular communication during infection. However, there is limited knowledge on whether EVs derived from M. pneumoniae-infected macrophages play as intercellular messengers and functional mechanisms. In this study, we establish a cell model of M. pneumoniae-infected macrophages that continuously secrete EVs to further asses their role as intercellular messengers and their functional mechanisms. Based on this model, we determined a method for isolating the pure EVs from M. pneumoniae-infected macrophages, which employs a sequence of operations, including differential centrifugation, filtering, and ultracentrifugation. We identified EVs and their purity using multiple methods, including electron microscopy, nanoparticle tracking analysis, Western blot, bacteria culture, and nucleic acid detection. EVs from M. pneumoniae-infected macrophages are pure, with a 30-200 nm diameter. These EVs can be taken up by uninfected macrophages and induce the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-8 through the nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPK) signals pathway. Moreover, the expression of inflammatory cytokines induced by EVs relies on TLR2-NF-κB/JNK signal pathways. These findings will help us better understand a persistent inflammatory response and cell-to-cell immune modulation in the context of M. pneumoniae infection.

The Universally Conserved Unconventional G Protein YchF Is Critical for Growth and Stress Response.

The ancient guanine nucleotide-binding (G) proteins are a group of critical regulatory and signal transduction proteins, widely involved in diverse cellular processes of all kingdoms of life. YchF is a kind of universally conserved novel unconventional G protein that appears to be crucial for growth and stress response in eukaryotes and bacteria. YchF is able to bind and hydrolyze both adenine nucleoside triphosphate (ATP) and guanosine nucleoside triphosphate (GTP), unlike other members of the P-loop GTPases. Hence, it can transduce signals and mediate multiple biological functions by using either ATP or GTP. YchF is not only a nucleotide-dependent translational factor associated with the ribosomal particles and proteasomal subunits, potentially bridging protein biosynthesis and degradation, but also sensitive to reactive oxygen species (ROS), probably recruiting many partner proteins in response to environmental stress. In this review, we summarize the latest insights into how YchF is associated with protein translation and ubiquitin-dependent protein degradation to regulate growth and maintain proteostasis under stress conditions.

Critical Role of 6-Phosphogluconate Dehydrogenase in TAp73-Mediated Cancer Cell Proliferation.

Cancer cells frequently alter their metabolism to support biogenesis and proliferation and survive specific metabolic stressors. The glucose-associated pentose phosphate pathway (PPP) is crucial for cancer cell proliferation. In particular, 6-phosphogluconate dehydrogenase (6PGD), the second dehydrogenase in the PPP, catalyzes the decarboxylation of 6-phosphogluconate into ribulose 5-phosphate (Ru5P). However, the mechanisms controlling 6PGD expression in cancer cells remain unclear. Herein, we show that TAp73 increases Ru5P and NADPH production through 6PGD activation to counteract reactive oxygen species and protects cells from apoptosis. Moreover, 6PGD overexpression rescues the proliferation and tumorigenic ability of TAp73-deficient cells. These findings further establish the critical role of TAp73 on glucose metabolism regulation, demonstrating that TAp73 can activate 6PGD expression to support oncogenic cell growth. IMPLICATIONS: By transcriptional upregulation of 6PGD, TAp73 promotes the generation of Ru5P and NADPH, and enhances tumor cell proliferation.

A Stability Training Method of Legged Robots Based on Training Platforms and Reinforcement Learning with Its Simulation and Experiment.

This paper continues the proposed idea of stability training for legged robots with any number of legs and any size on a motion platform and introduces the concept of a learning-based controller, the global self-stabilizer, to obtain a self-stabilization capability in robots. The overall structure of the global self-stabilizer is divided into three modules: action selection, adjustment calculation and joint motion mapping, with corresponding learning algorithms proposed for each module. Taking the human-sized biped robot, GoRoBoT-II, as an example, simulations and experiments in three kinds of motions were performed to validate the feasibility of the proposed idea. A well-designed training platform was used to perform composite random amplitude-limited disturbances, such as the sagittal and lateral tilt perturbations (±25°) and impact perturbations (0.47 times the robot gravity). The results show that the proposed global self-stabilizer converges after training and can dynamically combine actions according to the system state. Compared with the controllers used to generate the training data, the trained global self-stabilizer increases the success rate of stability verification simulations and experiments by more than 20% and 15%, respectively.

Qualitative and quantitative characterization of adsorption mechanisms for Cd2+ by silicon-rich biochar.

The adsorption characteristics of rice-husk biochar (RHB) rich in silicon (Si) for Cd2+ in solution and soil were investigated. Three biochars were produced at different pyrolytic temperatures of 300 °C(RHB300), 500 °C(RHB500) and 700 °C (RHB700). The pH effect, adsorption kinetics and isotherms were examined, and chemical analyses of Cd2+-loaded biochars were conducted by SEM-EDS, FTIR, XRD and Boehm titration. Biochars produced at higher temperature have much larger pH and surface area, resulting in greater adsorption capacities and faster adsorption kinetics. Maximum adsorption capacities calculated from Langmuir isotherm were 62.75, 77.37 and 93.50 mg/g for RHB300, RHB500 and RHB700, respectively. Cd2+ adsorption was primarily attributed to cation exchange and precipitation, which jointly contributed 59.55% (RHB300) to 76.05% (RHB700) of the total adsorption, but the mechanisms of complexation and coordination were of minor importance in total adsorption. The relationship of each mechanism with biochar's properties was further discussed. Si-containing minerals within biochar made a much larger contribution to precipitation than total adsorption, as the respective contribution proportion were 33.92% and 8.33% on average. When added to highly Cd-polluted soil, the biochars could effectively reduce the availability of Cd2+ after incubation for 35 days, and ameliorate soil acidification through the speediness of Si released into soil solutions. These demonstrate that rice husk-derived biochar, produced at high temperatures, can be suitable applied to mitigate Cd-contamination of soil and water, and the presented analyses shed light on the mechanisms underlying the adsorption by this Si-rich biochar.

Ionomic and metabolomic analyses reveal the resistance response mechanism to saline-alkali stress in Malus halliana seedlings.

Saline-alkali stress is a major abiotic stress limiting plant growth. The selection of saline-alkali-tolerant rootstock is an effective strategy to reduce salinization-alkalization influence in apple production. M. halliana is a highly saline-alkali-resistant apple rootstock in northwestern China. However, few metabolic response studies have been conducted on this species. In plants under saline-alkali stress, the uptake of K, Mg and Zn in M. halliana leaves were inhibited, whereas the absorption of Fe2+, Cu2+ or Mn2+ were increased. Metabolic analysis revealed 140 differentially expressed metabolites, which were mainly involved in alkaloid biosynthesis, phenylalanine biosynthesis, ATP-binding cassette (ABC) transporters, and mineral absorption. Especially, the expression of sucrose, amino acids, alkaloids, flavonoids and carotenoids were significantly upregulated under saline-alkali stress. qRT-PCR analysis demonstrated that NHX8 and ZTP1 involved in Na+ and Fe2+ transport were upregulated, while AKT1, MRS2-4 and ZTP29 involved in K+, Mg2+ and Zn2+ transport were downregulated, respectively. ANT, ATP2A, CALM and SOS2 are involved in Ca2+ signal transduction, and ABCB1, ABCC10 and NatA are key transporters that maintain ionic homeostasis. M. halliana regulates Na+/K+ homeostasis by mediating Ca2+ signalling and ABC transporters. The accumulation of metabolites contributes to improving the saline-alkali resistance of M. halliana because of the scavenging of ROS. An increase in pheophorbide a content in porphyrin and chlorophyll metabolism leads to leaf senescence in M. halliana leaves, which contributes to a reduction in stress-induced injury. These findings provide important insights into the saline-alkali tolerance mechanism in apple, which also provides an important starting point for future research.

Fabrication and properties of an injectable sodium alginate/PRP composite hydrogel as a potential cell carrier for cartilage repair.

Three-dimensional scaffolds like hydrogels can be employed as cell carriers for in vitro or in vivo colonization and have become a major research topic to replace damaged tissue. In the current study, a novel composite hydrogel composed of sodium alginate (SA) and platelet-rich-plasma (PRP) varying in blending ratios, cross-linked with calcium ions, released from calcium carbonate-D-Glucono-d-lactone (CaCO3 -GDL) was successfully prepared. It was found that addition of PRP changed largely the physical properties and biological performance of the composite hydrogels, which was depending on the blending ratio. The gelation rate and swelling ratio of alginate hydrogels were significantly reduced by the addition of PRP, which produced also a more homogeneous gel structure. Field emission scanning electron microscopy (FE-SEM) investigation confirmed the incorporation of PRP-derived proteins in the hydrogel, where a porous structure with a pore size of 200-300 µm was found. On the other hand, an increase in surface roughness was observed after the addition of PRP. The compressive mechanical strength of SA/PRP composite hydrogel was enhanced in comparison to the pure SA gel. The composite hydrogels with the highest PRP content exhibited at a maximum compressive stress of 0.26 MPa a maximum strain of 55%, while the maximum compressive strain of pure SA hydrogels was only 45% at a stress of 0.08 MPa. It was also found that the in vitro degradation of the alginate gel was accelerated by the addition of PRP. In terms of cellular responses, all gels exhibited an excellent cytocompatibility. Indeed, the composite hydrogels supported bone marrow-derived mesenchymal stem cells proliferation and their chondrogenesis with up-regulation of chondrogenic marker genes Sox9 and Aggrecan. Overall, the present study suggests a great potential of SA/PRP composite hydrogels as cell carriers for cartilage tissue engineering.

Relative distribution of Cd2+ adsorption mechanisms on biochars derived from rice straw and sewage sludge.

Qualitative and quantitative characterization of Cd2+ adsorption mechanisms was performed with rice-straw and sewage-sludge biochars produced at different temperature (300-700 °C), respectively. The pH effect, adsorption kinetics and isotherms were investigated, and chemical analyses of Cd2+-loaded biochars were conducted by SEM-EDS, XRD, FTIR and Boehm titration. This demonstrated that rice-straw biochars (RSBs) have greater adsorption capacities for Cd2+ than sewage-sludge biochars (SSBs), which was mainly due to precipitation and cation exchange mechanisms, with their contribution proportion to total adsorption from 76.1% to 80.8%. While in SSBs, both mechanisms were overshadowed by coordination with π electrons mechanism accounting for 59.2%-62.9% of total adsorption, even the role of cation exchange was negligible in the adsorption mechanisms accounting for 2.3%-6.7%. The relationship of each mechanism with biochar's properties were discussed, which further deepen our understanding of adsorption on biochars. These results suggest RSBs have great potential for removing Cd2+ from aqueous solutions.

Quantitative contribution of Cd2+ adsorption mechanisms by chicken-manure-derived biochars.

This study investigated the efficiency and mechanisms of Cd2+ removal by chicken-manure biochar produced at different temperatures. Adsorption kinetics, isotherms, thermodynamic, and desorption were examined, and the biochars before and after adsorption were analyzed by SEM-EDS, FTIR, Boehm titration, and XRD. Kinetics of adsorption were better described by pseudo-second-order kinetic model than pseudo-first-order kinetic and intraparticle diffusion model under different initial Cd2+ concentrations of 20, 50, and 100 mg L-1. Equilibrium adsorption was better modeled by Freundlich and Temkin isotherm equations than Langmuir equation at different temperatures of 25, 35, and 45 °C. Thermodynamic parameters confirmed the spontaneous and endothermic nature of the adsorption of Cd2+ at all of temperatures. Moreover, functional group complexation, precipitation, and cation exchange jointly contributed to Cd2+ adsorption on the biochars, whose relationship with the properties of biochar were also analyzed. The new precipitate as Cd5(PO4)3OH was found during the adsorption. Complexation and precipitation were predominant mechanisms for all biochars (together accounting for 92.4-98.8%), while cation exchange made a relatively minor contribution to total Cd2+ removal (accounting for 1.2-7.6%). The relative distribution of each mechanism on the biochars was determined, which deepen our understanding of the Cd2+ adsorption process. These results are useful for future practical applications of biochar to removal heavy metals from water.

Wnt/ß-catenin signaling pathway inhibits the proliferation and apoptosis of U87 glioma cells via different mechanisms.

The Wnt signaling pathway is necessary for the development of the central nervous system and is associated with tumorigenesis in various cancers. However, the mechanism of the Wnt signaling pathway in glioma cells has yet to be elucidated. Small-molecule Wnt modulators such as ICG-001 and AZD2858 were used to inhibit and stimulate the Wnt/ß-catenin signaling pathway. Techniques including cell proliferation assay, colony formation assay, Matrigel cell invasion assay, cell cycle assay and Genechip microarray were used. Gene Ontology Enrichment Analysis and Gene Set Enrichment Analysis have enriched many biological processes and signaling pathways. Both the inhibiting and stimulating Wnt/ß-catenin signaling pathways could influence the cell cycle, moreover, reduce the proliferation and survival of U87 glioma cells. However, Affymetrix expression microarray indicated that biological processes and networks of signaling pathways between stimulating and inhibiting the Wnt/ß-catenin signaling pathway largely differ. We propose that Wnt/ß-catenin signaling pathway might prove to be a valuable therapeutic target for glioma.

Glycogen synthase kinase 3 (GSK3)-inhibitor SB216763 promotes the conversion of human umbilical cord mesenchymal stem cells into neural precursors in adherent culture.

The ability to generate neural progenitor cells from human umbilical cord mesenchymal stem cells (hUC-MSCs) has provided an option to treat neurodegenerative diseases. To establish a method for this purpose, we characterized the early neural markers of hUC-MSCs-derived cells under different conditions. We found that neither the elimination of signals for alternative fate nor N2 supplement was sufficient to differentiate hUC-MSCs into neural precursor cells, but the GSK3 inhibitor SB216763 could promote an efficient neural commitment of hUC-MSCs. The results indicated that Wnt/ß-catenin might play an important role during the early neural differentiation of hUC-MSCs. Here, we report a method for hUC-MSCs to commit efficiently into a neural fate within a short period of time. This protocol provides an efficient method for hUC-MSCs-based neural regeneration.

Poly (L-lactic acid) porous scaffold-supported alginate hydrogel with improved mechanical properties and biocompatibility.

PURPOSE: Polymer porous scaffolds and hydrogels have been separately employed and explored for a wide range of applications including cell encapsulation, drug delivery, and tissue engineering. METHODS: In this study, a three-dimensional poly (L-lactic acid) (PLLA) scaffold with interconnected and homogeneously distributed pores was fabricated to support the alginate hydrogel (Alg). The gels were filled into the porous scaffold, which acted as an analogue of native extracellular matrix (ECM) for entrapment of cells within a support of predefined shape. The mechanical strength of the composite scaffold was characterized by compression testing. The chondrocyte behavior in the scaffold was determined by inverted microscopy, scanning electron microscopy (SEM) and MTT viability assay. The repair efficiency of such a composite scaffold was further investigated in dog spinal defects by histological evaluation after implantation for 4 weeks. RESULTS: Results showed that the composite scaffold possessed superior mechanical properties and hierarchical porous structure in comparison to pure Alg. Cell culture revealed that the cells presented a specific cartilage status in the composite scaffold in line with higher adherence and proliferation ratio. The histological analyses suggested that the composite scaffold substantially promotes its integration in the host tissue accompanied with a low inflammatory reaction and new tissue formation. CONCLUSIONS: The method thus provides a useful pathway for scaffold preparation that can simultaneously achieve suitable mechanical properties and good biocompatibility.

Inhibition of glycogen synthase kinase-3 (GSK3) promotes the neural differentiation of full-term amniotic fluid-derived stem cells towards neural progenitor cells.

The amniotic fluid has a heterogeneous population of cells. Some human amniotic fluid-derived stem (hAFS) cells have been shown to harbor the potential to differentiate into neural cells. However, the neural differentiation efficiency of hAFS cells remains low. In this study, we isolated CD117-positive hAFS cells from amniotic fluid and then examined the pluripotency of these cells through the formation of embryoid bodies (EBs). Additionally, we induced the neural differentiation of these cells using neuroectodermal medium. This study revealed that the GSK3-beta inhibitor SB216763 was able to stimulate the proliferation of CD117-positive hAFS cells without influencing their undifferentiated state. Moreover, SB216763 can efficiently promote the neural differentiation of CD117-positive hAFS cells towards neural progenitor cells in the presence of DMEM/F12 and N2 supplement. These findings provide an easy and low-cost method to maintain the proliferation of hAFS cells, as well as induce an efficacious generation of neural progenitor cells from hAFS cells. Such induction of the neural commitment of hAFS cells may provide an option for the treatment of neurodegenerative diseases by hAFS cells-based therapies.

Neural Commitment of Embryonic Stem Cells through the Formation of Embryoid Bodies (EBs)

An embryonic stem cell (ESC) is a good tool to generate neurons in vitro and can be used to mimic neural development in vivo. It has been widely used in research to examine the role of cell signalling during neuronal development, test the effects of drugs on neurons, and generate a large population of functional neurons. So far, a number of protocols have been established to promote the differentiation of ESCs, such as direct and indirect differentiation. One of the widely used protocols to generate neurons is through the spontaneous formation of multicellular aggregates known as embryonic bodies (EBs). However, for some, it is not clear why EB protocol could be the protocol of choice. EB also is known to mimic an early embryo; hence, knowing the similarities between EB and an early embryo is essential, particularly the information on the players that promote the formation of EBs or the aggregation of ESCs. This review paper focuses on these issues and discusses further the generation of neural cells from EBs using a well-known protocol, the 4−/4+ protocol.