Antitumor effects of Cypaliuruside F from Cyclocarya paliurus on HepG2 cells.

An undescribed dammarane triterpenoid saponin Cypaliuruside F was isolated from the leaves of Cyclocarya paliurus in our preliminary study. The MTT assay, flow cytometry, cell scratch, and DAPI staining were used to detect the antitumor effects of Cypaliuruside F on HepG2 cells. Subsequently, network pharmacology and molecular docking analysis were used to analyse the key targets of Cypaliuruside F against HCC. In addition, a Western blot was performed to determine the effects of Cypaliuruside F on the expression of key proteins in HepG2 cells. The experimental results indicated that the damarane triterpenoid saponin Cypaliuruside F from Cyclocarya paliurus inhibits the proliferation of HepG2 cells by inducing apoptosis and cell cycle arrest. These changes may promote the apoptosis of HepG2 cells by inhibiting the expression of mTOR, STAT3, and Bcl-2 while activating Bax.

AZD1390, an ataxia telangiectasia mutated inhibitor, attenuates microglia-mediated neuroinflammation and ischemic brain injury.

AIMS: Excessive neuroinflammation mediated mainly by microglia plays a crucial role in ischemic stroke. AZD1390, an ataxia telangiectasia mutated (ATM) specific inhibitor, has been shown to promote radio-sensitization and survival in central nervous system malignancies, while the role of AZD1390 in ischemic stroke remains unknown. METHODS: Real-time PCR, western blot, immunofluorescence staining, flow cytometry and enzyme-linked immunosorbent assays were used to assess the activation of microglia and the release of inflammatory cytokines. Behavioral tests were performed to measure neurological deficits. 2,3,5-Triphenyltetrazolium chloride staining was conducted to assess the infarct volume. The activation of NF-κB signaling pathway was explored through immunofluorescence staining, western blot, co-immunoprecipitation and proximity ligation assay. RESULTS: The level of pro-inflammation cytokines and activation of NF-κB signaling pathway was suppressed by AZD1390 in vitro and in vivo. The behavior deficits and infarct size were partially restored with AZD1390 treatment in experimental stroke. AZD1390 restrict ubiquitylation and sumoylation of the essential regulatory subunit of NF-κB (NEMO) in an ATM-dependent and ATM-independent way respectively, which reduced the activation of the NF-κB pathway. CONCLUSION: AZD1390 suppressed NF-κB signaling pathway to alleviate ischemic brain injury in experimental stroke, and attenuated microglia activation and neuroinflammation, which indicated that AZD1390 might be an attractive agent for the treatment of ischemic stroke.

Wwl70-induced ABHD6 inhibition attenuates memory deficits and pathological phenotypes in APPswe/PS1dE9 mice.

Synaptic dysfunction plays a crucial role in the pathogenesis of Alzheimer's disease (AD). α/ß-hydrolase domain-containing 6 (ABHD6) contributes to synaptic dysfunctions, and ABHD6 inhibition has shown potential therapeutic value in neurological disorders. However, the role of ABHD6 in AD has not been fully defined. In this study, we demonstrated that adeno-associated virus (AAV) mediated shRNA targeting ABHD6 in hippocampal neurons attenuated synaptic dysfunction and memory impairment of APPswe/PS1dE9 (APP/PS1) mice, while it didn't affect the amyloid-beta (Aß) levels and neuroinflammation in the brains. In addition, intraperitoneal injection of wwl70, a specific inhibitor of ABHD6, improved synaptic plasticity and memory function in APP/PS1 mice, which might attribute to the activation of endogenous cannabinoid signaling. Furthermore, wwl70 significantly decreased the Aß levels and neuroinflammation in the hippocampus of AD mice, and enhanced Aß phagocytized by microglia. In conclusion, for the first time our data have shown that ABHD6 inhibition might be a promising strategy for AD treatment, and wwl70 is a potential candidate for AD drug development pipeline.

Vascular invasion and lymph node metastasis mediate the effect of CA242 on prognosis in hilar cholangiocarcinoma patients after radical resection.

Background: Carbohydrate antigen 242 has been clinically used as a diagnostic biomarker for pancreatic cancer. However, the prognostic role of CA242 in hilar cholangiocarcinoma (HCCA) has not been identified. Also, it remains unclear to what extents the vascular invasion and lymph node metastasis mediate the effect of serum CA242 on prognosis. Objective: This study aimed to investigate whether vascular invasion and lymph node metastasis mediate the relationship between CA242 levels and clinical prognosis in HCCA patients after radical resection. Methods: Data of 234 HCCA patients who accepted radical resection from March 2008 to December 2014 were analyzed. Vascular invasion and lymph node metastasis were assessed by postoperative pathological examinations. Mediation analysis was performed to study the potential causal relationship between CA242 and overall survival (OS) and relapse-free survival (RFS). Survival analysis was performed using the Kaplan-Meier method. Results: Among 234 HCCA patients, 104 patients (44.4%) with normal CA242 levels (≤ 20 IU/ml) had significantly better OS (p=0.004) and RFS (p=0.001) than those 130 patients (55.6%) with elevated CA242 levels (>20 IU/ml). The logistic analysis showed that elevated CA242 was an independent risk factor for vascular invasion (p=0.006) and lymph nodes metastasis (p=0.040). The causal mediation analysis indicated that the vascular invasion (p=0.012 for OS; p=0.036 for RFS) and lymph nodes metastasis (p=0.024 for OS; p=0.014 for RFS) played significant roles in mediating the effect of serum CA242 on OS and RFS. Conclusion: Serum elevated CA242 could be a novel marker for prognosis prediction in HCCA patients. Vascular invasion and lymph node metastasis mediated the relationship between CA242 and clinical prognosis.

Transcriptomic Profile Identifies Hippocampal Sgk1 as the Key Mediator of Ovarian Estrogenic Regulation on Spatial Learning and Memory and Aß Accumulation.

Previous studies have shown that ovarian estrogens are involved in the occurrence and pathology of Alzheimer's disease (AD) through regulation on hippocampal synaptic plasticity and spatial memory; however, the underlying mechanisms have not yet been elucidated at the genomic scale. In this study, we established the postmenopausal estrogen-deficient model by ovariectomy (OVX). Then, we used high-throughput Affymetrix Clariom transcriptomics and found 143 differentially expressed genes in the hippocampus of OVX mice with the absolute fold change ≥ 1.5 and P < 0.05. GO analysis showed that the highest enrichment was seen in long-term memory. Combined with the response to steroid hormone enrichment and GeneMANIA network prediction, the serum and glucocorticoid-regulated kinase 1 gene (Sgk1) was found to be the most potent candidate for ovarian estrogenic regulation. Sgk1 overexpression viral vectors (oSgk1) were then constructed and injected into the hippocampus of OVX mice. Morris water maze test revealed that the impaired spatial learning and memory induced by OVX was rescued by Sgk1 overexpression. Additionally, the altered expression of synaptic proteins and actin remodeling proteins and changes in CA1 spine density and synapse density induced by OVX were also significantly reversed by oSgk1. Moreover, the OVX-induced increase in Aß-producing BACE1 and Aß and the decrease in insulin degrading enzyme were significantly reversed by oSgk1. The above results show that multiple pathways and genes are involved in ovarian estrogenic regulation of the function of the hippocampus, among which Sgk1 may be a novel potent target against estrogen-sensitive hippocampal dysfunctions, such as Aß-initiated AD.

Steroid receptor coactivator-1: The central intermediator linking multiple signals and functions in the brain and spinal cord.

The effects of steroid hormones are believed to be mediated by their nuclear receptors (NRs). The p160 coactivator family, including steroid receptor coactivator-1 (SRC-1), 2 and 3, has been shown to physically interact with NRs to enhance their transactivational activities. Among which SRC-1 has been predominantly localized in the central nervous system including brain and spinal cord. It is not only localized in neurons but also detectable in neuroglial cells (mainly localized in the nuclei but also detectable in the extra-nuclear components). Although the expression of SRC-1 is regulated by many steroids, it is also regulated by some non-steroidal factors such as injury, sound and light. Functionally, SRC-1 has been implied in normal function such as development and ageing, learning and memory, central regulation on reproductive behaviors, motor and food intake. Pathologically, SRC-1 may play a role in the regulation of neuropsychiatric disorders (including stress, depression, anxiety, and autism spectrum disorder), metabolite homeostasis and obesity as well as tumorigenesis. Under most conditions, the related mechanisms are far from elucidation; although it may regulate spatial memory through Rictor/mTORC2-actin polymerization related synaptic plasticity. Several inhibitors and stimulator of SRC-1 have shown anti-cancer potentials, but whether these small molecules could be used to modulate ageing and central disorder related neuropathology remain unclear. Therefore, to elucidate when and how SRC-1 is turned on and off under different stimuli is very interesting and great challenge for neuroscientists.

Mechanisms Underlying the Inhibition of KV1.3 Channel by Scorpion Toxin ImKTX58.

Voltage-gated KV1.3 channel has been reported to be a drug target for the treatment of autoimmune diseases, and specific inhibitors of Kv1.3 are potential therapeutic drugs for multiple diseases. The scorpions could produce various bioactive peptides that could inhibit KV1.3 channel. Here, we identified a new scorpion toxin polypeptide gene ImKTX58 from the venom gland cDNA library of the Chinese scorpion Isometrus maculatus Sequence alignment revealed high similarities between ImKTX58 mature peptide and previously reported KV1.3 channel blockers-LmKTX10 and ImKTX88-suggesting that ImKTX58 peptide might also be a KV1.3 channel blocker. By using electrophysiological recordings, we showed that recombinant ImKTX58 prepared by genetic engineering technologies had a highly selective inhibiting effect on KV1.3 channel. Further alanine scanning mutagenesis and computer simulation identified four amino acid residues in ImKTX58 peptide as key binding sites to KV1.3 channel by forming hydrogen bonds, salt bonds, and hydrophobic interactions. Among these four residues, 28th lysine of the ImKTX58 mature peptide was found to be the most critical amino acid residue for blocking KV1.3 channel. SIGNIFICANCE STATEMENT: In this study, we discovered a scorpion toxin gene ImKTX58 that has not been reported before in Hainan Isometrus maculatus and successfully used the prokaryotic expression system to express and purify the polypeptides encoded by this gene. Electrophysiological experiments on ImKTX58 showed that ImKTX58 has a highly selective blocking effect on KV1.3 channel over Kv1.1, Kv1.2, Kv1.5, SK2, SK3, and BK channels. These findings provide a theoretical basis for designing highly effective KV1.3 blockers to treat autoimmune and other diseases.

Parallel Optimization of Potency and Pharmacokinetics Leading to the Discovery of a Pyrrole Carboxamide ERK5 Kinase Domain Inhibitor.

The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.

Antidepressant-Like Effect and Mechanism of Ginsenoside Rd on Rodent Models of Depression.

Background: There is growing evidence to suggest that ginsenoside Rd (GRd) has a therapeutic effect on depression, but the specific mechanisms behind its activity require further study. Objective: This study is designed to investigate the antidepressant-like effect and underlying mechanisms of GRd. Methods: In this study, the behavioral despair mouse model of depression and chronic unpredictable mild stress (CUMS) rat model of depression were established to explore the effects of GRd on depression-like behavior and its underlying mechanisms. Behavioral tests were used to evaluate the replication of animal models and depression-like behaviors. The hypoxia-inducible factor-1α (HIF-1α) blocker 2-methoxyestradiol (2-ME) was injected to determine the role of HIF-1α in the antidepressant-like effect of GRd. In addition, molecular biology techniques were used to determine the mRNA and protein expression of HIF-1ɑ signaling pathway and synaptic plasticity-related regulators, that is synapsin 1 (SYN 1) and postsynaptic density protein 95 (PSD 95). In silico binding interaction studies of GRd with focused target proteins were performed using molecular docking to predict the affinity and optimal binding mode between ligands and receptors. Results: Our data show that GRd significantly reversed depression-like behavior and promoted mRNA and protein expression of HIF-1ɑ signaling pathway and synaptic plasticity-related regulators. However, the antidepressant-like effect of GRd disappeared upon inhibition of HIF-1α expression following administration of 2-ME. Furthermore, molecular docking results showed that GRd possessed significant binding affinity for HIF-1α, VEGF, and VEGFR-2. Conclusion: Our results show that GRd exhibits significant antidepressant-like effect and that HIF-1α signaling pathway is a promising target for the treatment of depression.

Long-term environmental cadmium exposure induced serum metabolic changes related to renal and liver dysfunctions in a female cohort from Southwest China.

Cadmium (Cd), a toxic heavy mental, has been reported to be correlated with increased incidences of multiple diseases. Only a few studies have paid attention to screen the urine metabolites related to long-term environmental Cd exposure in humans. Research on the Cd exposure-related serum metabolic alternations and biological mechanisms linking Cd exposure to adverse health risks in humans is scanty. In this study, we investigated the serum Cd exposure-related metabolic alternations in a cohort of 101 non-smoking females (two polluted groups and one control group) and 18 Cd exposure-related metabolites were identified. A total of 16 clinical indicators of renal and hepatic functions and bone health were measured. Five health effect biomarkers including serum creatinine, alkaline phosphatase, total bilirubin, direct bilirubin and albumin to globulin ratio that are related to impaired renal and hepatic functions showed significant differences among the three groups and had close correlations with Cd levels. We identified intermediate metabolites that were associated with both Cd exposure and health effect biomarkers using a "meet-in-the-middle" approach. Fourteen Cd exposure-related metabolites in the metabolism of glycerophospholipids, sphingolipids, arachidic acid, linoleic acid and amino acids, were identified to be the intermediates of Cd exposure and the health effect biomarkers. Our findings provided evidence for the linkage of long-term environmental Cd exposure and the renal and hepatic insufficiency.

[Analysis of volatile oil components of different species of Curcumae Rhizoma based on GC-MS and chemometrics].

The volatile oil of Curcumae Rhizoma has many active components,which are the key to the quality of Curcumae Rhizoma. Exploring the difference between volatile oil of different kinds of Curcumae Rhizoma facilitates the quality control and rational application of resources. In this study,GC-MS was applied to realize online qualitative and semi-quantitative analysis of the chemical composition spectrum of volatile oil from Curcuma wenyujin( CW),C. phaeocaulis( CP),and C. kwangsiensis( CK). Forty components were identified and their fingerprints were compared and evaluated. Hierarchical cluster analysis( HCA),principal component analysis( PCA),and orthogonal partial least squares discrimination analysis( OPLS-DA) were adopted to analyze the overall and outlier data. The results showed that the whole data could be divided into three kinds according to each analysis mode,and the volatile components of Curcumae Rhizoma vary greatly among species. PCA explored the difference between outliers and the mean value of the group and found that some volatile oils from CW may be greatly affected by the origin. By OPLS-DA,the samples from Zhejiang were able to gather,but those from Guizhou remained isolated,indicating the influence of growing environment on Curcumae Rhizoma metabolites. Based on VIP results combined with the heat map,characteristic volatile oil components of Curcumae Rhizoma from different varieties were screened out: curdione and linalool for CW; 2-undecanone for CP; humulene,γ-selinene,and zederone for CK. The GCMS method established in this study describes Curcumae Rhizoma samples comprehensively and accurately,and the characteristic components screened based on chemometrics can be used to distinguish Curcumae Rhizoma from different varieties and give them unique pharmacodynamic significance,which is fast,convenient,stable,and reliable and supports the rational application of Curcu-mae Rhizoma resources. It is found that the region of origin has great influence on CW,which is worthy of further study.

Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in p27KIP1 Degradation.

The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.

Co-exposure of cadmium and lead on bone health in a southwestern Chinese population aged 40-75 years.

Both cadmium (Cd) and lead (Pb) are associated with bone health, but studies exploring the effects of Cd and Pb co-exposure on bone health are rare. This study aimed to assess the interactive effects of Cd and Pb co-exposure on bone health. In total, 799 participants, living in the targeted areas (located in southwestern China) for more than 15 years, aged 40-75 years, and subsisted on homegrown rice and vegetables were investigated. Cd and Pb levels in urine and blood samples, as well as bone mineral density, T- and Z-score were determined. After being adjusted for covariates, the T-score was negatively correlated with blood Pb in men (P < .05); for women and non-smoking women, the T-score was negatively correlated with urinary Pb (P < .05). Moreover, after being adjusted for covariates, the Z-score was negatively correlated with urinary Pb in non-smoking women (P < .05). No positive association of prevalence of osteoporosis with Cd and Pb exposure was found. However, at an additive scale, positive interactions of urinary Cd and Pb on the prevalence of osteoporosis for women and non-smoking women, and the same interactions to blood Cd and Pb for men were found. There was also a positive interaction of urinary Cd and Pb for women at a multiplicative scale. This study suggests Cd and Pb exposure could exert detrimental effects on bone health, with possible underlying interactions. Nevertheless, more studies are needed to explore the interactive effects of heavy metal co-exposure.

Identification of a novel orally bioavailable ERK5 inhibitor with selectivity over p38α and BRD4.

Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82 µM for ERK5; IC50 > 120 µM for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.

Hippocampus-specific Rictor knockdown inhibited 17ß-estradiol induced neuronal plasticity and spatial memory improvement in ovariectomized mice.

Estrogens have been shown to play profound roles in the regulation of the structure and function of the hippocampus; however, the underlying mechanism is not clear. Previous studies have shown that when Rictor, the core component of the mammalian target of the rapamycin complex 2 (mTORC2), was deleted, hippocampal actin polymerization was reduced and long-term memory was seriously impaired. Although hippocampal Rictor could be regulated by estrogen receptor agonists/antagonists, whether Rictor could directly mediate estrogenic regulation of neuronal plasticity, spatial learning and memory remains unclear. In this study, we first examined the regulation of hippocampal Rictor and P-AKTser473 (P-AKT) by E2, then we used Rictor-specific dsRNA (shRictor) injected into the hippocampi of E2-treated ovariectomized (OVX) mice or into cultured cells. The results showed that both Rictor and P-AKT could be regulated by E2. OVX induced actin depolymerization, decreases in CA1 spine density and synapse density as well as changes in synaptic proteins were reversed by E2 replacement. However, these E2-mediated effects were significantly blocked by shRictor treatment. Similar results were also demonstrated by in vitro cell culture studies using E2 and/or shRictor. Importantly, we found that E2 replacement induced improvements in learning and memory impairment seen in OVX mice were significantly blocked by shRictor. Taken together, the current studies provided the first direct evidence for the important role of Rictor in estrogenic action on the hippocampus, indicating that it may be a therapeutic target for the treatment of E2-related, hippocampus-dependent cognitive dysfunction.

Differences in the Conformational Energy Landscape of CDK1 and CDK2 Suggest a Mechanism for Achieving Selective CDK Inhibition.

Dysregulation of the cell cycle characterizes many cancer subtypes, providing a rationale for developing cyclin-dependent kinase (CDK) inhibitors. Potent CDK2 inhibitors might target certain cancers in which CCNE1 is amplified. However, current CDK2 inhibitors also inhibit CDK1, generating a toxicity liability. We have used biophysical measurements and X-ray crystallography to investigate the ATP-competitive inhibitor binding properties of cyclin-free and cyclin-bound CDK1 and CDK2. We show that these kinases can readily be distinguished by such inhibitors when cyclin-free, but not when cyclin-bound. The basis for this discrimination is unclear from either inspection or molecular dynamics simulation of ligand-bound CDKs, but is reflected in the contacts made between the kinase N- and C-lobes. We conclude that there is a subtle but profound difference between the conformational energy landscapes of cyclin-free CDK1 and CDK2. The unusual properties of CDK1 might be exploited to differentiate CDK1 from other CDKs in future cancer therapeutic design.

Dietary cadmium exposure assessment in rural areas of Southwest China.

Dietary exposure of cadmium (Cd) has not been studied in Southwest China. The objective of the study was to determine the pollution characteristics and contamination levels in various agriculture products in Southwest China and to conduct a comparison of dietary exposure assessment of Cd in polluted and non-polluted areas. Results showed that the mean Cd contents in rice were 0.53 and 0.52 mg/kg in the high-polluted and low-polluted areas, respectively, with the average value was 0.03 mg/kg in the control area. The mean dietary Cd exposure from rice and vegetables of the selected non-occupational residents in Southwest China was 113.10 µg/kg bodyweight (bw)/month, 88.80 µg/kg bw/month, and 16.50 µg/kg bw/month in the high-polluted, low-polluted, and control areas, respectively, which correspond to 4.5 times, 3.6 times, and 0.66 times of the provisional tolerable monthly intake (25 µg/kg bw/month) established by the Joint FAO/WHO Expert Committee on Food Additives. The findings indicated that the risk for Cd exposure of residents was high due to home-grown food (most especially rice) being near polluted areas and is of great concern.

Antitumor activity of fucoidan in anaplastic thyroid cancer via apoptosis and anti-angiogenesis.

The present study demonstrated the effect of fucoidan, isolated from Fucus vesiculosus, on cell growth and apoptosis in anaplastic thyroid cancer cells. The cell viability was analyzed using a Cell Counting Kit­8 cell proliferation kit. Diamidino-2-phenylindole and terminal deoxynucleotidyl transferase-mediated dUTP nick­end labeling assays were used to examine the apoptotic effect of fucoidan, which revealed the presence of apoptotic bodies and DNA fragmentation. Fucoidan inhibited the growth of FTC133 and TPC1 ATC cells in a dose­dependent manner. It also induced the apoptosis of FTC133 cells by promoting the expression levels of cleaved poly ADP­ribose polymerase and caspase­3. Significant decreases in the levels expression of hypoxia-inducible factor 1α and vascular endothelial growth factor were observed in the FTC133 cells following treatment of the cells with fucoidan. In addition, inhibition in tube formation and the migration of FTC133 cells were observed in the cells treated with fucoidan, compared with the cells in the control group. Therefore, fucoidan inhibited cell growth, induced apoptosis and suppressed angiogenesis in the thyroid cancer cells.

Long-term propagation of tree shrew spermatogonial stem cells in culture and successful generation of transgenic offspring.

Tree shrews have a close relationship to primates and have many advantages over rodents in biomedical research. However, the lack of gene manipulation methods has hindered the wider use of this animal. Spermatogonial stem cells (SSCs) have been successfully expanded in culture to permit sophisticated gene editing in the mouse and rat. Here, we describe a culture system for the long-term expansion of tree shrew SSCs without the loss of stem cell properties. In our study, thymus cell antigen 1 was used to enrich tree shrew SSCs. RNA-sequencing analysis revealed that the Wnt/ß-catenin signaling pathway was active in undifferentiated SSCs, but was downregulated upon the initiation of SSC differentiation. Exposure of tree shrew primary SSCs to recombinant Wnt3a protein during the initial passages of culture enhanced the survival of SSCs. Use of tree shrew Sertoli cells, but not mouse embryonic fibroblasts, as feeder was found to be necessary for tree shrew SSC proliferation, leading to a robust cell expansion and long-term culture. The expanded tree shrew SSCs were transfected with enhanced green fluorescent protein (EGFP)-expressing lentiviral vectors. After transplantation into sterilized adult male tree shrew's testes, the EGFP-tagged SSCs were able to restore spermatogenesis and successfully generate transgenic offspring. Moreover, these SSCs were suitable for the CRISPR/Cas9-mediated gene modification. The development of a culture system to expand tree shrew SSCs in combination with a gene editing approach paves the way for precise genome manipulation using the tree shrew.