Dynamic culture of cerebral organoids using a pillar/perfusion plate for the assessment of developmental neurotoxicity.

Despite the potential toxicity of commercial chemicals to the development of the nervous system (known as developmental neurotoxicity or DNT), conventional in vitro cell models have primarily been employed for the assessment of acute neuronal toxicity. On the other hand, animal models used for the assessment of DNT are not physiologically relevant due to the heterogenic difference between humans and animals. In addition, animal models are low-throughput, time-consuming, expensive, and ethically questionable. Recently, human brain organoids have emerged as a promising alternative to assess the detrimental effects of chemicals on the developing brain. However, conventional organoid culture systems have several technical limitations including low throughput, lack of reproducibility, insufficient maturity of organoids, and the formation of the necrotic core due to limited diffusion of nutrients and oxygen. To address these issues and establish predictive DNT models, cerebral organoids were differentiated in a dynamic condition in a unique pillar/perfusion plate, which were exposed to test compounds to evaluate DNT potential. The pillar/perfusion plate facilitated uniform, dynamic culture of cerebral organoids with improved proliferation and maturity by rapid, bidirectional flow generated on a digital rocker. Day 9 cerebral organoids in the pillar/perfusion plate were exposed to ascorbic acid (DNT negative) and methylmercury (DNT positive) in a dynamic condition for 1 and 3 weeks, and changes in organoid morphology and neural gene expression were measured to determine DNT potential. As expected, ascorbic acid didn't induce any changes in organoid morphology and neural gene expression. However, exposure of day 9 cerebral organoids to methylmercury resulted in significant changes in organoid morphology and neural gene expression. Interestingly, methylmercury did not induce adverse changes in cerebral organoids in a static condition, thus highlighting the importance of dynamic organoid culture in DNT assessment.

GV1001 reduces neurodegeneration and prolongs lifespan in 3xTg-AD mouse model through anti-aging effects.

GV1001, which mimics the activity of human telomerase reverse transcriptase, protects neural cells from amyloid beta (Aß) toxicity and other stressors through extra-telomeric function, as noted in our prior in vitro studies. As per a recent phase II clinical trial, it improves cognitive function in patients with moderate to severe dementia. However, the underlying protective mechanisms remain unclear. This study aimed to investigate the effects of GV1001 on neurodegeneration, senescence, and survival in triple transgenic Alzheimer's disease (3xTg-AD) mice. GV1001 (1 mg/kg) was subcutaneously injected into old 3xTg-AD mice thrice a week until the endpoint for sacrifice, and survival was analysed. Magnetic resonance imaging (MRI) and Prussian blue staining (PBS) were performed to evaluate entry of GV1001 entrance into the brain. Diverse molecular studies were performed to investigate the effect of GV1001 on neurodegeneration and cellular senescence in AD model mice, with a particular focus on BACE, amyloid beta1-42 (Aß1-42), phosphorylated tau, volume of dentate gyrus, ß-galactosidase positive cells, telomere length, telomerase activity, and ageing-associated proteins. GV1001 crossed the blood-brain barrier, as confirmed by assessing the status of ferrocenecarboxylic acid-conjugated GV1001 using magnetic resonance imaging and PBS. GV1001 increased the survival of 3xTg-AD mice. It decreased BACE and Aß1-42 levels, neurodegeneration (i.e., reduced CA1, CA3 and dentate gyrus volume, decreased levels of senescence-associated ß-galactosidase positive cells, and increased telomere length and telomerase activity), and levels of ageing-associated proteins. We suggest that GV1001 exerts anti-ageing effects in 3xTg-AD mice by reducing neurodegeneration and senescence, which contributes to improved survival.

Uniform cerebral organoid culture on a pillar plate by simple and reproducible spheroid transfer from an ultralow attachment well plate.

Human induced pluripotent stem cell (iPSC)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effectivein vitromodel for studying both normal brain development and disorders. However, current brain organoid culture methods face several challenges, including low throughput, high variability in organoid generation, and time-consuming, multiple transfer and encapsulation of cells in hydrogels throughout the culture. These limitations hinder the widespread application of brain organoids including high-throughput assessment of compounds in clinical and industrial lab settings. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies in an ultra-low attachment 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95%-100%. Using this approach, we robustly generated cerebral organoids on the pillar plate and demonstrated an intra-batch coefficient of variation below 9%-19% based on ATP-based cell viability and compound treatment. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowingin situorganoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

Brain organoids: A revolutionary tool for modeling neurological disorders and development of therapeutics.

Brain organoids are self-organized, three-dimensional (3D) aggregates derived from pluripotent stem cells that have cell types and cellular architectures resembling those of the developing human brain. The current understanding of human brain developmental processes and neurological disorders has advanced significantly with the introduction of this in vitro model. Brain organoids serve as a translational link between two-dimensional (2D) cultures and in vivo models which imitate the neural tube formation at the early and late stages and the differentiation of neuroepithelium with whole-brain regionalization. In addition, the generation of region-specific brain organoids made it possible to investigate the pathogenic and etiological aspects of acquired and inherited brain disease along with drug discovery and drug toxicity testing. In this review article, we first summarize an overview of the existing methods and platforms used for generating brain organoids and their limitations and then discuss the recent advancement in brain organoid technology. In addition, we discuss how brain organoids have been used to model aspects of neurodevelopmental and neurodegenerative diseases, including autism spectrum disorder (ASD), Rett syndrome, Zika virus-related microcephaly, Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD).

GV1001 modulates neuroinflammation and improves memory and behavior through the activation of gonadotropin-releasing hormone receptors in a triple transgenic Alzheimer's disease mouse model.

GV1001 protects neural cells from amyloid-ß (Aß) toxicity and other stressors in in vitro studies and demonstrates clinically beneficial effects in patients with moderate to severe Alzheimer's disease (AD). Here, we investigated the protective effects and mechanism of action of GV1001 in triple transgenic AD (3xTg-AD) mice. We found that GV1001 improved memory and cognition in middle- and old-aged 3xTg-AD mice. Additionally, it reduced Aß oligomer and phospho-tau (Ser202 and Thr205) levels in the brain, and mitigated neuroinflammation by promoting a neuroprotective microglial and astrocyte phenotype while diminishing the neurotoxic ones. In vitro, GV1001 bound to gonadotropin releasing hormone receptors (GnRHRs) with high affinity. Levels of cyclic adenosine monophosphate, a direct downstream effector of activated GnRHRs, increased after GV1001 treatment. Furthermore, inhibition of GnRHRs blocked GV1001-induced effects. Thus, GV1001 might improve cognitive and memory functions of 3xTg-AD mice by suppressing neuroinflammation and reducing Aß oligomers levels and phospho-tau by activating GnRHRs and their downstream signaling pathways.

Uniform cerebral organoid culture on a pillar plate by simple and reproducible spheroid transfer from an ultralow attachment well plate.

Human induced pluripotent stem cell (iPSCs)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effective in vitro model for studying both normal brain development and disorders. However, current brain organoid culture methods face several challenges, including low throughput, high variability in organoid generation, and time-consuming, multiple transfer and encapsulation of cells in hydrogels throughout the culture. These limitations hinder the widespread application of brain organoids including high-throughput assessment of compounds in clinical and industrial lab settings. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies (EBs) in an ultra-low attachment (ULA) 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95 - 100%. By differentiating the EBs on the pillar plate, we achieved robust generation of cerebral organoids with a coefficient of variation (CV) below 19%. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowing in situ organoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

SOX7: Novel Autistic Gene Identified by Analysis of Multi-Omics Data.

Background: Despite thousands of variants identified by genome-wide association studies (GWAS) to be associated with autism spectrum disorder (ASD), it is unclear which mutations are causal because most are noncoding. Consequently, reliable diagnostic biomarkers are lacking. RNA-seq analysis captures biomolecular complexity that GWAS cannot by considering transcriptomic patterns. Therefore, integrating DNA and RNA testing may reveal causal genes and useful biomarkers for ASD. Methods: We performed gene-based association studies using an adaptive test method with GWAS summary statistics from two large Psychiatric Genomics Consortium (PGC) datasets (ASD2019: 18,382 cases and 27,969 controls; ASD2017: 6,197 cases and 7,377 controls). We also investigated differential expression for genes identified with the adaptive test using an RNA-seq dataset (GSE30573: 3 cases and 3 controls) and DESeq2. Results: We identified 5 genes significantly associated with ASD in ASD2019 (KIZ-AS1, p = 8.67×10- 10; KIZ, p = 1.16×10- 9; XRN2, p = 7.73×10- 9; SOX7, p = 2.22×10- 7; LOC101929229 (also known as PINX1-DT), p = 2.14×10- 6). Two of the five genes were replicated in ASD2017: SOX7 (p = 0.00087) and LOC101929229 (p = 0.009), and KIZ was close to the replication boundary of replication (p = 0.06). We identified significant expression differences for SOX7 (p = 0.0017, adjusted p = 0.0085), LOC101929229 (p = 5.83×10- 7, adjusted p = 1.18×10- 5), and KIZ (p = 0.00099, adjusted p = 0.0055). SOX7 encodes a transcription factor that regulates developmental pathways, alterations in which may contribute to ASD. Limitations: The limitation of the gene-based analysis is the reliance on a reference population for estimating linkage disequilibrium between variants. The similarity of this reference population to the population of study is crucial to the accuracy of many gene-based analyses, including those performed in this study. As a result, the extent of our findings is limited to European populations, as this was our reference of choice. Future work includes a tighter integration of DNA and RNA information as well as extensions to non-European populations that have been under-researched. Conclusions: These findings suggest that SOX7 and its related SOX family genes encode transcription factors that are critical to the downregulation of the canonical Wnt/ß-catenin signaling pathway, an important developmental signaling pathway, providing credence to the biologic plausibility of the association between gene SOX7 and autism spectrum disorder.

Dyeing Performance and Anti-Superbacterial Activity of Cotton Fabrics Dyed with Chamaecyparis obtusa.

In hospitals, doctors' and patients' uniforms, as well as bedding and textiles, can be carriers of superbacteria. This study was conducted to test the anti-superbacterial activity of cotton fabrics dyed with extracts of Chamaecyparis obtusa (C. obtusa). The dye was extracted by boiling C. obtusa in water. The test cotton was mordant-dyed three times with the solution at a 1:17 dyeing bath ratio and at an 8.69% (o.w.f) dye concentration for 15 min at 40 °C. C. obtusa dyeing demonstrated a high dyeing affinity in the absence of mordant (K/S value = 14.62). The K/S value of the dyed fabric increased in the order of Cu-mordanted, Fe-mordanted, non-mordanted, and Al-mordanted cotton. Dry cleaning, perspiration and rubbing fastness were determined to be good (Grade 4-5). The dyed fabrics appeared to have a high deodorizing ability compared to the control fabric. They showed not only antibacterial activity against Staphylococcus aureus (S. aureus) and Klebsiella pneumoniae (K. pneumoniae), known to be frequently found in fabrics, but also higher antibacterial activity against the superbacteria methicillin-resistant Staphylococcus aureus (MRSA) (reduced by 99.7%). These results suggest that fabric dyed with C. obtusa extract may be used in clothes and bed linens for inpatients, given its high anti-superbacterial activity. Furthermore, such fabrics may contribute to inhibiting pathogenic infections when used in hospital uniforms or operation gowns for doctors or nurses in hospitals.

SOX7: Novel Autistic Gene Identified by Analysis of Multi-Omics Data.

Background: Genome-wide association studies and next generation sequencing data analyses based on DNA information have identified thousands of mutations associated with autism spectrum disorder (ASD). However, more than 99% of identified mutations are non-coding. Thus, it is unclear which of these mutations might be functional and thus potentially causal variants. Transcriptomic profiling using total RNA-sequencing has been one of the most utilized approaches to link protein levels to genetic information at the molecular level. The transcriptome captures molecular genomic complexity that the DNA sequence solely does not. Some mutations alter a gene's DNA sequence but do not necessarily change expression and/or protein function. To date, few common variants reliably associated with the diagnosis status of ASD despite consistently high estimates of heritability. In addition, reliable biomarkers used to diagnose ASD or molecular mechanisms to define the severity of ASD do not exist. Objectives: It is necessary to integrate DNA and RNA testing together to identify true causal genes and propose useful biomarkers for ASD. Methods: We performed gene-based association studies with adaptive test using genome-wide association studies (GWAS) summary statistics with two large GWAS datasets (ASD 2019 data: 18,382 ASD cases and 27,969 controls [discovery data]; ASD 2017 data: 6,197 ASD cases and 7,377 controls [replication data]) which were obtained from the Psychiatric Genomics Consortium (PGC). In addition, we investigated differential expression for genes identified in gene-based GWAS with a RNA-seq dataset (GSE30573: 3 cases and 3 controls) using the DESeq2 package. Results: We identified 5 genes significantly associated with ASD in ASD 2019 data (KIZ-AS1, p=8.67×10-10; KIZ, p=1.16×10-9; XRN2, p=7.73×10-9; SOX7, p=2.22×10-7; PINX1-DT, p=2.14×10-6). Among these 5 genes, gene SOX7 (p=0.00087), LOC101929229 (p=0.009), and KIZ-AS1 (p=0.059) were replicated in ASD 2017 data. KIZ (p=0.06) was close to the boundary of replication in ASD 2017 data. Genes SOX7 (p=0.0017, adjusted p=0.0085), LOC101929229 (also known as PINX1-DT, p=5.83×10-7, adjusted p=1.18×10-5), and KIZ (p=0.00099, adjusted p=0.0055) indicated significant expression differences between cases and controls in the RNA-seq data. SOX7 encodes a member of the SOX (SRY-related HMG-box) family of transcription factors pivotally contributing to determining of the cell fate and identity in many lineages. The encoded protein may act as a transcriptional regulator after forming a protein complex with other proteins leading to autism. Conclusion: Gene SOX7 in the transcription factor family could be associated with ASD. This finding may provide new diagnostic and therapeutic strategies for ASD.

Inhibition of the NLRP3 Inflammasome Activation/Assembly through the Activation of the PI3K Pathway by Naloxone Protects Neural Stem Cells from Ischemic Condition.

Naloxone is a well-known opioid antagonist and has been suggested to have neuroprotective effects in cerebral ischemia. We investigated whether naloxone exhibits anti-inflammatory and neuroprotective effects in neural stem cells (NSCs) injured by oxygen-glucose deprivation (OGD), whether it affects the NOD-like receptor protein 3 (NLRP3) inflammasome activation/assembly, and whether the role of the phosphatidylinositol 3-kinase (PI3K) pathway is important in the control of NLRP3 inflammasome activation/assembly by naloxone. Primary cultured NSCs were subjected to OGD and treated with different concentrations of naloxone. Cell viability, proliferation, and the intracellular signaling proteins associated with the PI3K pathway and NLRP3 inflammasome activation/assembly were evaluated in OGD-injured NSCs. OGD significantly reduced survival, proliferation, and migration and increased apoptosis of NSCs. However, treatment with naloxone significantly restored survival, proliferation, and migration and decreased apoptosis of NSCs. Moreover, OGD markedly increased NLRP3 inflammasome activation/assembly and cleaved caspase-1 and interleukin-1ß levels in NSCs, but naloxone significantly attenuated these effects. These neuroprotective and anti-inflammatory effects of naloxone were eliminated when cells were treated with PI3K inhibitors. Our results suggest that NLRP3 inflammasome is a potential therapeutic target and that naloxone reduces ischemic injury in NSCs by inhibiting NLRP3 inflammasome activation/assembly mediated by the activation of the PI3K signaling pathway.

Homogeneity of XEN Cells Is Critical for Generation of Chemically Induced Pluripotent Stem Cells.

In induced pluripotent stem cells (iPSCs), pluripotency is induced artificially by introducing the transcription factors Oct4, Sox2, Klf4, and c-Myc. When a transgene is introduced using a viral vector, the transgene may be integrated into the host genome and cause a mutation and cancer. No integration occurs when an episomal vector is used, but this method has a limitation in that remnants of the virus or vector remain in the cell, which limits the use of such iPSCs in therapeutic applications. Chemical reprogramming, which relies on treatment with small-molecule compounds to induce pluripotency, can overcome this problem. In this method, reprogramming is induced according to the gene expression pattern of extra-embryonic endoderm (XEN) cells, which are used as an intermediate stage in pluripotency induction. Therefore, iPSCs can be induced only from established XEN cells. We induced XEN cells using small molecules that modulate a signaling pathway and affect epigenetic modifications, and devised a culture method in which can be produced homogeneous XEN cells. At least 4 passages were required to establish morphologically homogeneous chemically induced XEN (CiXEN) cells, whose properties were similar to those of XEN cells, as revealed through cellular and molecular characterization. Chemically iPSCs derived from CiXEN cells showed characteristics similar to those of mouse embryonic stem cells. Our results show that the homogeneity of CiXEN cells is critical for the efficient induction of pluripotency by chemicals.

Inhibition of Class I Histone Deacetylase Enhances Self-Reprogramming of Spermatogonial Stem Cells into Pluripotent Stem Cells.

Background and Objectives: Spermatogonial stem cells (SSCs) are the most primitive cells in spermatogenesis and are the only adult stem cells capable of passing on the genome of a given species to the next generation. SSCs are the only adult stem cells known to exhibit high Oct4 expression and can be induced to self-reprogram into pluripotent cells depending on culture conditions. Epigenetic modulation is well known to be involved in the induction of pluripotency of somatic cells. However, epigenetic modulation in self-reprogramming of SSCs into pluripotent cells has not been studied. Methods and Results: In this study, we examined the involvement of epigenetic modulation by assessing whether self-reprogramming of SSCs is enhanced by treatment with epigenetic modulators. We found that second-generation selective class I HDAC inhibitors increased SSC reprogramming efficiency, whereas non-selective HDAC inhibitors had no effect. Conclusions: We showed that pluripotent stem cells derived from adult SSCs by treatment with small molecules with epigenetic modulator functions exhibit pluripotency in vitro and in vivo. Our results suggest that the mechanism of SSC reprogramming by epigenetic modulator can be used for important applications in epigenetic reprogramming research.

Pinus koraiensis Essential Oil Attenuates the Pathogenicity of Superbacteria by Suppressing Virulence Gene Expression.

In the quest to combat infections attributable to antibiotic-resistant superbacteria, an essential oil derived from the needles of Pinus koraiensis Sieb. et Zucc. (PKEO) has emerged as a promising solution. In this study, we demonstrate that PKEO can be used to inhibit the growth, glucose metabolite acidogenicity, and biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA). Quantitative PCR analysis provided direct evidence that PKEO reduces the mRNA expression of the accessory gene regulator A (agrA) and staphylococcal accessory regulator A (sarA), thereby indicating its inhibitory effect on pathogenic regulatory genes. Chromatographic analyses of PKEO identified terpene hydrocarbons as prominent essential oil constituents. These compounds, notably α-pinene, limonene, and ß-caryophyllene, have been established to have antimicrobial properties. Our findings indicate that an oil derived from P. koraiensis can effectively combat antibiotic-resistant strains by disrupting the pathogenicity regulatory system, thereby establishing PKEO as a promising candidate for the treatment of MRSA infections.

Enhancement of proliferation of human umbilical cord blood-derived CD34+ hematopoietic stem cells by a combination of hyper-interleukin-6 and small molecules.

Umbilical cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells (HSCs) for transplantation to treat various hematological disorders. The major limitation to the use of UCB-derived HSCs (UCB-HSCs) in transplantation, however, is the low numbers of HSCs in a unit of cord blood. To overcome this limitation, various cytokines or small molecules have been used to expand UCB-HSCs ex vivo. In this study, we investigated a synergistic effect of the combination of HIL-6, SR1, and UM171 on UCB-HSC culture and found that this combination resulted in the highest number of CD34+ cells. These results suggest that the combination of SR1, UM171 and HIL-6 exerts a synergistic effect in the proliferation of HSCs from UCB and thus, SR1, UM171 and HIL-6 is the most suitable combination for obtaining HSCs from UCB for clinical transplantation.

CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar.

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1-sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

Establishment of an integration-free human induced pluripotent stem cell line (TJCi001-A) from normal bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) possess excellent therapeutic potential for the treatment of various diseases including graft-versus-host disease, rheumatoid arthritis, osteoarthritis, and multiple sclerosis. Here, we generated an induced pluripotent stem cell (iPSC) line from BM-MSCs employing a non-integrating episomal vector. The generated iPSCs expressed pluripotency markers, showed a normal karyotype, and exhibited the potential for in vitro differentiation into three germ layers. This iPSC line can be used as a healthy control in stem cell therapeutics and disease modeling studies.

Proteomic Analysis Reveals Commonly Secreted Proteins of Mesenchymal Stem Cells Derived from Bone Marrow, Adipose Tissue, and Synovial Membrane to Show Potential for Cartilage Regeneration in Knee Osteoarthritis.

Paracrine factors secreted by mesenchymal stem cells (MSCs) reportedly modulate inflammation and reparative processes in damaged tissues and have been explored for knee osteoarthritis (OA) therapy. Although various studies have reported the effects of paracrine factors in knee OA, it is not yet clear which paracrine factors directly affect the regeneration of damaged cartilage and which are secreted under various knee OA conditions. In this study, we cultured MSCs derived from three types of tissues and treated each type with IL-1ß and TNF-α or not to obtain conditioned medium. Each conditioned medium was used to analyse the paracrine factors related to cartilage regeneration using liquid chromatography-tandem mass spectrometry. Bone marrow-, adipose tissue-, and synovial membrane-MSCs (all-MSCs) exhibited expression of 93 proteins under normal conditions and 105 proteins under inflammatory conditions. It was confirmed that the types of secreted proteins differed depending on the environmental conditions, and the proteins were validated using ELISA. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis using a list of proteins secreted by all-MSCs under each condition confirmed that the secreted proteins were closely related to cartilage repair under inflammatory conditions. Protein-protein interaction networks were confirmed to change depending on environmental differences and were found to enhance the secretion of paracrine factors related to cartilage regeneration under inflammatory conditions. In conclusion, our results demonstrated that compared with knee OA conditions, the differential expression proteins may contribute to the regeneration of damaged cartilage. In addition, the detailed information on commonly secreted proteins by all-MSCs provides a comprehensive basis for understanding the potential of paracrine factors to influence tissue repair and regeneration in knee OA.

Gui-A-Gra Attenuates Testicular Dysfunction in Varicocele-Induced Rats via Oxidative Stress, ER Stress and Mitochondrial Apoptosis Pathway.

Gui-A-Gra, a commercial insect powder from Gryllus bimaculatus, is registered as an edible insect by the Korean food and drug administration. The aim of this study was to investigate the effect of Gui-A-Gra on testicular damage induced by experimental left varicocele in male Sprague Dawley rats. A total of 72 rats were randomly divided into the following six groups (12 rats in each group): a normal control group (CTR), a group administrated with Gui-A-Gra 1.63 gm/kg (G1.63), a group administrated with Gui-A-Gra 6.5 gm/kg (G6.5), a varicocele (VC)-induced control group (VC), a VC-induced group administrated with Gui-A-Gra 1.63 gm/kg (VC + G1.63), and a VC-induced group administrated with Gui-A-Gra 6.5 gm/kg (VC + G6.5). Rats were administrated 1.63 or 6.5 gm/kg Gui-A-Gra once daily for 42 days. Indicators of sperm parameters, histopathology, reproductive hormones, oxidative stress, endoplasmic reticulum (ER) stress, inflammation, and mitochondrial apoptosis were analyzed to evaluate effects of Gui-A-Gra on VC-induced testicular dysfunction. Gui-A-Gra administration to VC-induced rats significantly (p < 0.05) increased sperm count and sperm motility, Johnsen score, spermatogenic cell density, serum testosterone, testicular superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, GPx4, and steroidogenic acute regulatory protein (StAR) level. Moreover, pretreatment with Gui-A-Gra significantly (p < 0.05) decreased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive cells/tubules, serum luteinizing hormone (LH), serum follicle-stimulating hormone (FSH), testicular tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), malondialdehyde (MDA), reactive oxygen species (ROS)/reactive nitrogen species (RNS) level, glucose-regulated protein-78 (Grp-78), phosphorylated c-Jun-N-terminal kinase (p-JNK), phosphorylated inositol-requiring transmembrane kinase/endoribonuclease 1α (p-IRE1α), cleaved caspase-3, and BCL2 associated X protein: B-cell lymphoma 2 (Bax: Bcl2) ratio in VC rats. These results suggest that protective effects of Gui-A-Gra on VC-induced testicular injury might be due to its antioxidant, anti-inflammatory, and androgenic activities that might be mediated via crosstalk of oxidative stress, ER stress, and mitochondrial apoptosis pathway.

Generation of OCT4-EGFP, NANOG-tdTomato dual reporter human induced pluripotent stem cell line, KKUi001-A, using the CRISPR/Cas9 system.

OCT4 and NANOG are core transcription factor genes in self-renewal, differentiation, and reprogramming. Here, we generated an OCT4-EGFP, NANOG-tdTomato dual reporter hiPSC line, KKUi001-A, on the basis of human induced pluripotent stem cells using CRISPR/Cas9 technology. EGFP and tdTomato reporter were inserted into before the stop codon of OCT4 and NANOG, respectively. Simultaneous expression of EGFP and tdTomato was observed when expression of OCT4 and NANOG was changed during differentiation and reprogramming. KKUi001-A hiPSC line will be a useful tool to find initial time point of OCT4 and NANOG expression during reprogramming process and to screen small molecules that promote reprogramming.

MOTILIPERM Ameliorates Immobilization Stress-Induced Testicular Dysfunction via Inhibition of Oxidative Stress and Modulation of the Nrf2/HO-1 Pathway in SD Rats.

It is well established that physiological stress has an adverse effect on the male reproductive system. Experimental studies have demonstrated the promising effects of MOTILIPERM in male infertility. MOTILIPERM extract is composed of three crude medicinal herbs: Morinda officinalis How (Rubiaceae) roots, Allium cepa L. (Liliaceae) outer scales, and Cuscuta chinensis Lamark (convolvulaceae) seeds. The present study aimed to investigate the possible mechanisms responsible for the effects of MOTILIPERM on testicular dysfunction induced by immobilization stress. Fifty male Sprague Dawley rats were divided into five groups (10 rats each): a normal control group (CTR), a control group administered MOTILIPERM 200 mg/kg (M 200), an immobilization-induced stress control group (S), an immobilization-induced stress group administered MOTILIPERM 100 mg/kg (S + M 100), and MOTILIPERM 200 mg/kg (S + M 200). Stressed rats (n = 30) were subjected to stress by immobilization for 6 h by placing them in a Perspex restraint cage, while controls (n = 20) were maintained without disturbance. Rats were administrated 100 or 200 mg/kg MOTILIPERM once daily for 30 days 1 h prior to immobilization. At the end of the treatment period, we measured body and reproductive organ weight; sperm parameters; histopathological damage; reproductive hormone levels; steroidogenic acute regulatory protein (StAR); biomarkers of oxidative stress; and apoptosis markers. MOTILIPERM treatment improved testicular dysfunction by up-regulating (p < 0.05) sperm count, sperm motility, serum testosterone level, StAR protein level, Johnsen score, and spermatogenic cell density in stressed rats. MOTILIPERM decreased oxidative stress by increasing (p < 0.05) testicular superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione peroxidase-4 (GPx 4), catalase, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) levels and decreasing (p < 0.05) malondialdehyde (MDA) and reactive oxygen species/reactive nitrogen species (ROS/RNS) levels. Furthermore, MOTILIPERM down-regulated (p < 0.05) cleaved caspase 3 and BCL2 associated X protein (Bax) levels; increased pro caspase-3 and B-cell lymphoma 2 (Bcl-2) levels; and upregulated testicular germ cell proliferation in stressed rats. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells and serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels also significantly (p < 0.05) decreased after pretreatment with MOTILIPERM in stressed rats. Collectively, our results suggest that, in immobilization-mediated stress-induced testicular dysfunction, MOTILIPERM sustains normal spermatogenesis via antioxidant and anti-apoptotic activities by activating the NRF/HO-1 signaling pathway.