Primary Cultures Research Articles

Cuiru Keli Improves Postpartum Hypogalactia in Rats Through Secreted Frizzled-Related Protein 2-Wnt/β-catenin Signaling Pathway

Based on the secreted frizzled-related protein 2 (SFRP2)-Wnt/β-catenin signaling pathway, this study explored the effect and mechanism of Cuiru Keli (CRKL) in the treatment of postpartum hypogalactia. A rat model of postpartum hypogalactia was established by gavaging 2 mL of 1.6 mg/mL bromocriptine mesylate to female rats on the third day after delivery. Female rats with a delivery time difference of less than 48 hours were selected and randomly assigned to 7 groups, including a normal group (without any modeling or medication), a model group, a CRKL low-dose group of model group model rats receiving CRKL at the dose of 3 g/kg, a CRKL medium-dose group of model rats receiving CRKL at the dose of 6 g/kg, a CRKL high-dose group of model rats receiving CRKL at the dose of 9 g/kg, a positive drug group of model rats receiving domperidone at the dose of 3 mg/kg, and a negative control (NC) group of model rats receiving normal saline. Each group contained 6 rats. Except for the normal and model groups, the remaining 5 groups were continuously administered with the respective intervention drugs at the specified doses by gavage once a day for 10 days. Changes in the total litter mass of the offspring in the 7 groups within 10 days were measured, and HE staining was performed to identify pathological changes in the mammary tissue (MT). Six groups of rats (excluding the positive control group) were used to observe the pathological changes of eosinophils in pituitary tissue. ELISA was performed to determine the content of prolactin (PRL) in serum, immunohistochemical staining was used to determine the expression of prolactin receptor (PRLR) in MT, and RT-qPCR was used to determine the mRNA expression of genes related to lactation in MT. Network pharmacology and molecular docking were used to study the therapeutic effect and mechanism of CRKL on postpartum hypogalactia, particularly whether it acted through the SFRP2-Wnt/β-catenin signaling pathway. The mechanism of CRKL treatment was further validated by detecting mRNA (RT-qPCR) and protein expression (Western blot) of related pathway genes. Cell experiments were conducted using primary culture rat mammary epithelial cells (RMEC) from rat MT. RMEC were divided into four groups, including a normal group (primary culture RMEC, untreated), SFRP2 overexpression group (primary cultured RMEC treated with SFRP2 overexpression vector), SFRP2 overexpression+CRKL group (receiving treatment for SFRP2 overexpression group plus 10% drug-containing serum), and negative control group (primary culture RMEC treated with empty vector). The effect of CRKL on the expression of lactation-related genes FASN, CSN2, and GLUT1 mRNA after SFRP2 overexpression was detected by RT-qPCR. In this study, CRKL was administered at a dose of 3 g/kg in the CRKL low-dose group, 6 g/kg in the medium-dose group, and 9 g/kg in the high-dose group (P<0.05 or P<0.01). Compared with the model group, CRKL at all doses significantly increased the total litter weight gain of the offsprings within 10 days (P<0.05 or P<0.01), and effectively increased lactation (P<0.01), the area of mammary lobules, and the size and filling of acinar cavities. CRKL at all doses also increased the number of eosinophils that secreted PRL in the pituitary gland of the postpartum hypogalactia rat model, and increased the content of PRL in the serum (P<0.05 or P<0.01). CRKL promoted the secretion and expression of PRL in postpartum hypogalactic model rats. In addition, it significantly promoted the expression of genes related to milk fat, milk protein, and lactose synthesis in MT (P<0.05 or P<0.01). Network pharmacology predicted that the Wnt signaling pathway might be a key pathway for CRKL in treating postpartum hypogalactia. The molecular docking results showed that related chemical components in CRKL had good binding ability with CCND1 and SFRP2. Compared with the model group, CRKL at all doses inhibited the expression of SFRP2 gene in vivo (P<0.01) and activated the mRNA and protein expression of CCND1 and c-Myc in the Wnt/β-catenin signaling pathway in MT (P<0.05 or P<0.01). Cell experiments showed that, compared to the normal group, SFRP2 overexpression reduced the mRNA expression of milk synthesis-related genes FASN, CSN2, and GLUT1 in RMEC (P<0.01). The CCK8 results indicated that 10% of the drug-containing serum was the effective concentration administered to cells (P<0.01). After administering drug-containing serum, the expression of the lactation-related genes FASN, CSN2, and GLUT1 were up-regulated (compared with the SFRP2 overexpression group, P<0.01). CRKL alleviates postpartum hypogalactia through the SFRP2-Wnt/β-catenin signaling pathway. SFRP2 might be a potential new target for the diagnosis and treatment of postpartum hypogalactia. This reveals a new mechanism of CRKL in treating postpartum hypogalactia and promotes its clinical application.

Cystatin C loaded in brain-derived extracellular vesicles rescues synapses after ischemic insult in vitro and in vivo

Synaptic loss is an early event in the penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. We aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse potential beneficial effects of these candidates for a prospective stroke treatment. For this, we performed Liquid Chromatography coupled to Mass Spectrometry (LC–MS)-based proteomics of synaptosomes isolated from the ipsilateral hemispheres of mice subjected to experimental stroke at different time points (24 h, 4 and 7 days) and compared them to sham-operated mice. Proteomic analyses indicated that, among the differentially expressed proteins between the two groups, cystatin C (CysC) was significantly increased at 24 h and 4 days following stroke, before returning to steady-state levels at 7 days, thus indicating a potential transient and intrinsic rescue mechanism attempt of neurons. When CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, this treatment significantly improved the preservation of synaptic structures. Notably, similar effects were observed when CysC was loaded into brain-derived extracellular vesicles (BDEVs). Finally, when CysC contained in BDEVs was administered intracerebroventricularly to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25, Homer-1, and NCAM in the penumbra area compared to the group supplied with empty BDEVs. Thus, we show that CysC-loaded BDEVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of a therapeutic use in stroke patients.

Palmitic Acid Exerts Anti-Tumorigenic Activities by Modulating Cellular Stress and Lipid Droplet Formation in Endometrial Cancer.

Epidemiological and clinical evidence have extensively documented the role of obesity in the development of endometrial cancer. However, the effect of fatty acids on cell growth in endometrial cancer has not been widely studied. Here, we reported that palmitic acid significantly inhibited cell proliferation of endometrial cancer cells and primary cultures of endometrial cancer and reduced tumor growth in a transgenic mouse model of endometrial cancer, in parallel with increased cellular stress and apoptosis and decreased cellular adhesion and invasion. Inhibition of cellular stress by N-acetyl-L-cysteine effectively reversed the effects of palmitic acid on cell proliferation, apoptosis, and invasive capacity in endometrial cancer cells. Palmitic acid increased the intracellular formation of lipid droplets in a time- and dose-dependent manner. Depletion of lipid droplets by blocking DGAT1 and DGAT2 effectively increased the ability of palmitic acid to inhibit cell proliferation and induce cleaved caspase 3 activity. Collectively, this study provides new insight into the effect of palmitic acid on cell proliferation and invasion and the formation of lipid droplets that may have potential clinical relevance in the treatment of obesity-driven endometrial cancer.

Astrocyte atrophy induced by L-PGDS/PGD2/Src signaling dysfunction in the central amygdala mediates postpartum depression

BackgroundPostpartum depression (PPD) is a serious psychiatric disorder that has significantly adverse impacts on maternal health. Metabolic abnormalities in the brain are associated with numerous neurological disorders, yet the specific metabolic signaling pathways and brain regions involved in PPD remain unelucidated. MethodsWe performed behavioral test in the virgin and postpartum mice. We used mass spectrometry imaging (MSI) and targeted metabolomics analyses to investigate the metabolic alternation in the brain of GABAAR Delta-subunit-deficient (Gabrd−/−) postpartum mice, a specific preclinical animal model of PPD. Next, we performed mechanism studies including qPCR, Western blot, immunofluorescence staining, electron microscopy and primary astrocyte culture. In the specific knockdown and rescue experiments, we injected the adeno-associated virus into the central amygdala (CeA) of female mice. ResultsWe identified that prostaglandin D2 (PGD2) downregulation in the CeA was the most outstanding alternation in PPD, and then validated that lipocalin-type prostaglandin D synthase (L-PGDS)/PGD2 downregulation plays a causal role in depressive behaviors derived from PPD in both wild-type and Gabrd−/− mice. Furthermore, we verified that L-PGDS/PGD2 signaling dysfunction-induced astrocytes atrophy is mediated by Src phosphorylation both in vitro and in vivo. LimitationsL-PGDS/PGD2 signaling dysfunction may be only responsible for the depressive behavior rather than maternal behaviors in the PPD, and it remains to be seen whether this mechanism is applicable to all depression types. ConclusionOur study identified abnormalities in the L-PGDS/PGD2 signaling in the CeA, which inhibited Src phosphorylation and induced astrocyte atrophy, ultimately resulting in the development of PPD in mice.

Leeches Baicalobdella torquata feed on hemolymph but have a low effect on the cellular immune response of amphipod Eulimnogammarus verrucosus from Lake Baikal.

Lake Baikal is one of the largest and oldest freshwater reservoirs on the planet with a huge endemic diversity of amphipods (Amphipoda, Crustacea). These crustaceans have various symbiotic relationships, including the rarely described phenomenon of leech parasitism on amphipods. It is known that leeches feeding on hemolymph of crustacean hosts can influence their physiology, especially under stressful conditions. Here we show that leeches Baicalobdella torquata (Grube, 1871) found on gills of Eulimnogammarus verrucosus (Gerstfeldt, 1858), one of the most abundant amphipods in the Baikal littoral zone, indeed feed on the hemolymph of their host. However, the leech infection had no effect on immune parameters such as hemocyte concentration or phenoloxidase activity and also did not affect glycogen content. The intensity of hemocyte reaction to foreign bodies in a primary culture was identical between leech-free and leech-infected animals. Artificial infection with leeches also had only a subtle effect on the course of a model microbial infection in terms of hemocyte concentration and composition. Despite we cannot fully exclude deleterious effects of the parasites, our study indicates a low influence of a few leeches on E. verrucosus and shows that leech-infected amphipods can be used at least for some types of ecophysiological experiments.

Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway

Oxidative stress-induced death of neurons and astrocytes contributes to the pathogenesis of numerous neurodegenerative diseases. While significant progress has been made in identifying neuroprotective molecules against neuronal oxidative damage, little is known about their counterparts for astrocytes. Prolactin (PRL), a hormone known to stimulate astroglial proliferation, viability, and cytokine expression, exhibits antioxidant effects in neurons. However, its role in protecting astrocytes from oxidative stress remains unexplored. Here, we investigated the effect of PRL against hydrogen peroxide (H2O2)-induced oxidative insult in primary cortical astrocyte cultures. Incubation of astrocytes with PRL led to increased enzymatic activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX), resulting in higher total antioxidant capacity. Concomitantly, PRL prevented H2O2-induced cell death, reactive oxygen species accumulation, and protein and lipid oxidation. The protective effect of PRL upon H2O2-induced cell death can be explained by the activation of both signal transducer and activator of transcription 3 (STAT3) and NFE2 like bZIP transcription factor 2 (NRF2) transduction cascades. We demonstrated that PRL induced nuclear translocation and transcriptional upregulation of Nrf2, concurrently with the transcriptional upregulation of the NRF2-dependent genes heme oxygenase 1, Sod1, Sod2, and Gpx1. Pharmacological blockade of STAT3 suppressed PRL-induced transcriptional upregulation of Nrf2, Sod1 and Gpx1 mRNA, and SOD and GPX activities. Furthermore, genetic ablation of the PRL receptor increased astroglial susceptibility to H2O2-induced cell death and superoxide accumulation, while diminishing their intrinsic antioxidant capacity. Overall, these findings unveil PRL as a potent antioxidant hormone that protects astrocytes from oxidative insult, which may contribute to brain neuroprotection.

Autonomous and Self-sustained Circadian Oscillators Displayed in Human Primary Parathyroid Cell Culture Drive Parathormone Secretion

Abstract Background Most cells in our body possess circadian oscillators controlling diurnal rhythmicity of body metabolism. The role of molecular clocks in regulating the function and disfunction of human parathyroid gland (PG), responsible for calcium homeostasis, has not been unraveled. Hyperparathyroidism is a common endocrine pathology, characterized by an unadapted PTH secretion to calcium levels. Aims Here we aimed at characterizing molecular makeup of circadian clocks in human primary parathyroid cell culture (HPPCC), and at establishing differential transcriptional patterns of normal and pathological PGs. Methods RNA extracted from normal, adenomatous, and hyperplastic PG tissues was subjected to RNA sequencing (RNAseq) analysis. The rim of normal PG tissue was dissected from parathyroid adenoma specimens based on the higher autofluorescence intensity assessed by the near infra-red imaging. HPPCCs were established from adenomatous and hyperplastic PG biopsies. Efficient circadian clock disruption was achieved in HPPCCs by small interfering RNA-mediated knockdown of CLOCK. The functionality of circadian clock machinery was assessed by continuous recording of circadian bioluminescence introduced via lentivectors and paralleled with around-the-clock measurement of PTH secretion using perfusion system. Results The RNAseq analyses revealed transcriptional signatures that distinguished between pathological and normal PGs, and among adenomatous and hyperplastic PGs. We report, for the first time, robust anti-phasic circadian rhythmicity of Per2-luciferase and Bmal1-luciferase reporters in HPPCCs synchronized in vitro. Strikingly, we observed circadian rhythmicity of PTH secretion by parathyroid adenoma cells. Moreover, clock disruption in PG adenoma cells has an impact on transcription of functional genes. Conclusion Our data indicate presence of cell-autonomous molecular clock in human PG cells paralleled with circadian rhythmic PTH secretion and provide large-scale transcriptional pattern of parathyroid adenoma and hyperplastic tissues.

Characterization of Japanese Encephalitis Virus Isolated from Persistently Infected Mouse Embryo Cells.

Japanese encephalitis virus (JEV) has a positive-sense single-stranded RNA genome and belongs to the genus Flavivirus of the family Flaviviridae. Persistent JEV infection was previously shown in pig blood cells, which act as a natural reservoir of this virus. We aimed to determine the pathogenicity factors involved in persistent JEV infection by analyzing the pathogenicity and genome sequences of a virus isolated from a persistent infection model. We established persistent JEV infections in cells by inoculating mouse fetus primary cell cultures with the Beijing-1 strain of JEV and then performing repeated infected cell passages, harvesting viruses after each passage while monitoring the plaque size over 100 generations. The virus growth rate was compared among Vero, C6/36, and Neuro-2a cells. The pathogenicity was examined in female ICR mice at several ages. Additionally, we determined the whole-genome sequences. The 134th Beijing-1-derived persistent virus (ME134) grew in Vero cells at a similar rate to the parent strain but did not grow well in C6/36 or Neuro-2a cells. No differences were observed in pathogenicity after intracerebral inoculation in mice of different ages, but the survival time was extended in older mice. Mutations in the persistent virus genomes were found across all regions but were mainly focused in the NS3, NS4b, and 3'NCR regions, with a 34-base-pair deletion found in the variable region. The short deletion in the 3'NCR region appeared to be responsible for the reduced pathogenicity and growth efficiency.

Reversibility of Endoplasmic Reticulum Stress Markers During Long-Term Glucose Starvation in Astrocytes

Previous studies have demonstrated a brain volume decrease linked to long-term starvation in patients with anorexia nervosa (AN). Food intake is critically diminished in this disorder, leading to one of the highest mortality rates within the psychiatric disease spectrum. As reported in animal models, astrocytes seem to be the most affected cell type in AN. In a recently established primary cell culture model, an elevated unfolded protein response (UPR) was observed in long-term glucose semi-starved astrocytes. A well-functioning protein machinery is essential for every cell, and prolonged UPR will lead to cell death. As a nucleic acid stress-sensing pathway with the activator located in the endoplasmic reticulum, the regulation of the cGAS-STING pathway (cyclic GMP-AMP synthase/stimulator of interferon genes) was additionally investigated in the starvation context. In the current study, a glucose semi-starvation protocol of 15 days, during which cells were supplied with 2 mM glucose in the medium, was prolonged with an additional 6-day long recovery period. Our findings showed that increased UPR mRNA expression was reversible after re-establishing the standard glucose concentration of 25 mM. Furthermore, we were able to verify the presence of cGAS and STING in astrocytes with a characteristic presence of cGAS in the astrocyte nucleus during starvation. A correlation between STING and the glial fibrillary acidic protein (GFAP) could be established, hinting at a conditional presence of STING with a specific astrocyte phenotype.Graphical

A Comparative Analysis of Secreted miRNAs Reveals Candidate Biomarkers for Pleural Mesothelioma Detection

Abstract Background MicroRNAs secreted by tumour cells through exosomes or in non-encapsulated form, were shown to promote tumour growth and treatment resistance, underscoring their potential as cancer biomarkers. Aims Here, we compare miRNAs secreted by pleural mesothelioma (PM) primary cells with miRNAs secreted by non-PM cells, in order to identify diagnostic biomarkers of this devastating disease. Methods We established primary cell cultures from pleural effusion of 12 PM and 7 non-PM patients. Secreted miRNAs were profiled as: (1) total secreted miRNA in cell culture supernatant (Sup), and (2) exosomal (Exo) miRNAs. Exosomes were extracted using iZON qEV columns. RNA was extracted with mirVana PARIS kit. Sequencing libraries were prepared using the QIAseq miRNA Library Kit (Qiagen). Reads were mapped on the mature miRNA sequences (MirBaseDB), followed by DESeq2 differential expression analysis. MiRNA target genes were selected using MirDB and functionally annotated using DAVID. Results We identified 309 and 84 deregulated miRNAs in PM-Exo and PM-Sup samples, respectively (p-value &amp;lt;0.05, Fig. 1A). Among the up-regulated miRNAs, we found let-7c-3p and miR-16-5p, which are known tumour suppressors, and miR-23a-3p and miR-30a-5p (Fig. 1B). Interestingly, miR-30a-5p belongs to the same family as miR-30e-5p, which together with miR-23a-3p was previously identified as part of the long survival signature in PM. Additionally, we identified 11 miRNAs that showed up-regulation in both PM-Exo and PM-Sup. Further analysis of these candidate miRNAs revealed that exosomal expression of miR-30a-5p was a significant predictor of patient survival (Fig. 1C). Finally, we identified potential target genes of the 11 up-regulated miRNAs - the target genes were significantly associated with transcriptional regulation and cell division (p-value &amp;lt;0.05). Conclusion Our comparative analysis of secreted miRNAs identified 11 candidate biomarkers for PM. Our findings emphasise the importance of the PM secretome in advancing our understanding of mesothelioma biology and discovering novel cell-free biomarkers. Further in-depth analyses are currently underway.

Foxg1 bimodally tunes L1-mRNA and -DNA dynamics in the developing murine neocortex.

Foxg1 masters telencephalic development via a pleiotropic control over its progression. Expressed within the central nervous system (CNS), L1 retrotransposons are implicated in progression of its histogenesis and tuning of its genomic plasticity. Foxg1 represses gene transcription, and L1 elements share putative Foxg1-binding motifs, suggesting the former might limit telencephalic expression (and activity) of the latter. We tested such a prediction, in vivo as well as in engineered primary neural cultures, using loss- and gain-of-function approaches. We found that Foxg1-dependent, transcriptional L1 repression specifically occurs in neopallial neuronogenic progenitors and post-mitotic neurons, where it is supported by specific changes in the L1 epigenetic landscape. Unexpectedly, we discovered that Foxg1 physically interacts with L1-mRNA and positively regulates neonatal neopallium L1-DNA content, antagonizing the retrotranscription-suppressing activity exerted by Mov10 and Ddx39a helicases. To the best of our knowledge, Foxg1 represents the first CNS patterning gene acting as a bimodal retrotransposon modulator, limiting transcription of L1 elements and promoting their amplification, within a specific domain of the developing mouse brain.

Advances in the study of gastric organoids as disease models.

Gastric organoids are models created in the laboratory using stem cells and sophisticated three-dimensional cell culture techniques. These models have shown great promise in providing valuable insights into gastric physiology and advanced disease research. This review comprehensively summarizes and analyzes the research advances in culture methods and techniques for adult stem cells and induced pluripotent stem cell-derived organoids, and patient-derived organoids. The potential value of gastric organoids in studying the pathogenesis of stomach-related diseases and facilitating drug screening is initially discussed. The construction of gastric organoids involves several key steps, including cell extraction and culture, three-dimensional structure formation, and functional expression. Simulating the structure and function of the human stomach by disease modeling with gastric organoids provides a platform to study the mechanism of gastric cancer induction by Helicobacter pylori. In addition, in drug screening and development, gastric organoids can be used as a key tool to evaluate drug efficacy and toxicity in preclinical trials. They can also be used for precision medicine according to the specific conditions of patients with gastric cancer, to assess drug resistance, and to predict the possibility of adverse reactions. However, despite the impressive progress in the field of gastric organoids, there are still many unknowns that need to be addressed, especially in the field of regenerative medicine. Meanwhile, the reproducibility and consistency of organoid cultures are major challenges that must be overcome. These challenges have had a significant impact on the development of gastric organoids. Nonetheless, as technology continues to advance, we can foresee more comprehensive research in the construction of gastric organoids. Such research will provide better solutions for the treatment of stomach-related diseases and personalized medicine.

Cannabinoid and Orexigenic Systems Interplay as a New Focus of Research in Alzheimer’s Disease

Alzheimer’s disease (AD) remains a significant health challenge, with an increasing prevalence globally. Recent research has aimed to deepen the understanding of the disease pathophysiology and to find potential therapeutic interventions. In this regard, G protein-coupled receptors (GPCRs) have emerged as novel potential therapeutic targets to palliate the progression of neurodegenerative diseases such as AD. Orexin and cannabinoid receptors are GPCRs capable of forming heteromeric complexes with a relevant role in the development of this disease. On the one hand, the hyperactivation of the orexins system has been associated with sleep–wake cycle disruption and Aβ peptide accumulation. On the other hand, cannabinoid receptor overexpression takes place in a neuroinflammatory environment, favoring neuroprotective effects. Considering the high number of interactions between cannabinoid and orexin systems that have been described, regulation of this interplay emerges as a new focus of research. In fact, in microglial primary cultures of APPSw/Ind mice model of AD there is an important increase in CB2R–OX1R complex expression, while OX1R antagonism potentiates the neuroprotective effects of CB2R. Specifically, pretreatment with the OX1R antagonist has been shown to strongly potentiate CB2R signaling in the cAMP pathway. Furthermore, the blockade of OX1R can also abolish the detrimental effects of OX1R overactivation in AD. In this sense, CB2R–OX1R becomes a new potential therapeutic target to combat AD.

Establishment of a new equine embryo brain primary cell culture with long-term expansion

Primary cell cultures derived from human embryo lung play a crucial role in virology by aiding virus propagation and vaccine development. These cultures exhibit a notable ability to undergo multiple subcultures, often reaching up to 70 passages. However, finding alternative primary cell cultures with similar longevity and usefulness is challenging. In this study, we introduce a novel primary culture cells derived from equine embryo brain (FEB), which cells exhibited remarkable long-term cultivation potential. The FEB was established and maintained using Sumitomo Nerve-Cell Culture System Comparison studies were conducted with fetal equine kidney cell line (FEK-Tc13) to assess growth rates and subculture longevity. Immunological characterization was performed using neuronal markers to confirm the neural nature of FEB cells. Viral growth assessments were conducted using equine herpesviruses (EHV-1 and EHV-4) to evaluate infectivity and cytopathic effects in FEB cells. PCR analysis and real-time PCR assays were employed to detect viral genomic DNA and transcription activity of EHVs in infected FEB cells. FEB cells demonstrated faster growth rates compared to fetal equine kidney cell line (FEK-Tc13 cells) and exhibited sustained subculture capability exceeding 50 passages. Immunostaining confirmed the glial identity of FEB cells. Both equine herpesviruses 1 and 4 EHV-1 and EHV-4 viruses efficiently replicated in FEB cells, resulting in clear cytopathic effects. PCR analysis detected genomic DNA of EHVs in infected FEB cells, indicating successful viral infection. The establishment of FEB cells with extended subculture capability highlights their potential utility as a model system for studying neural cell biology and viral infections.

Establishment, characterization, and genetic profiling of patient-derived osteosarcoma cells from a patient with retinoblastoma

Osteosarcoma is the most common malignant bone cancer in pediatric patients. Patients who respond poorly to chemotherapy experience worse clinical outcomes with a high mortality rate. The major challenge is the lack of effective drugs for these patients. To introduce new drugs for clinical approval, preclinical studies based on in vitro models must demonstrate the potency of the tested drugs, enabling the drugs to enter phase 1 clinical trials. Patient-derived cell culture is a promising testing platform for in vitro studies, as they more accurately recapitulate cancer states and genetic profiles compared to cell lines. In the present study, we established patient-derived osteosarcoma cells (PDC) from a patient who had previously been diagnosed with retinoblastoma. We identified a new variant of a germline mutation in the RB1 gene in the tissue of the patient. The biological effects of this PDC were studied to observe whether the cryopreserved PDC retained a feature of fresh PDC. The cryopreserved PDC preserved the key biological effects, including cell growth, invasive capability, migration, and mineralization, that define the conserved phenotypes compared to fresh PDC. From whole genome sequencing analysis of osteosarcoma tissue and patient-derived cells, we found that cryopreserved PDC was a minor population in the origin tissue and was selectively grown under the culture conditions. The cryopreserved PDC has a high resistance to conventional chemotherapy. This study demonstrated that the established cryopreserved PDC has the aggressive characteristics of osteosarcoma, in particular the chemoresistance phenotype that might be used for further investigation in the chemoresistant mechanism of osteosarcoma. In conclusion, the approach we applied for primary cell culture might be a promising method to generate in vitro models for functional testing of osteosarcoma.

Sulfated Hydrogels as Primary Intervertebral Disc Cell Culture Systems.

The negatively charged extracellular matrix plays a vital role in intervertebral disc tissues, providing specific cues for cell maintenance and tissue hydration. Unfortunately, suitable biomimetics for intervertebral disc regeneration are lacking. Here, sulfated alginate was investigated as a 3D culture material due to its similarity to the charged matrix of the intervertebral disc. Precursor solutions of standard alginate, or alginate with 0.1% or 0.2% degrees of sulfation, were mixed with primary human nucleus pulposus cells, cast, and cultured for 14 days. A 0.2% degree of sulfation resulted in significantly decreased cell density and viability after 7 days of culture. Furthermore, a sulfation-dependent decrease in DNA content and metabolic activity was evident after 14 days. Interestingly, no significant differences in cell density and viability were observed between surface and core regions for sulfated alginate, unlike in standard alginate, where the cell number was significantly higher in the core than in the surface region. Due to low cell numbers, phenotypic evaluation was not achieved in sulfated alginate biomaterial. Overall, standard alginate supported human NP cell growth and viability superior to sulfated alginate; however, future research on phenotypic properties is required to decipher the biological properties of sulfated alginate in intervertebral disc cells.

The AMPK activator ATX-304 alters cellular metabolism to protect against cisplatin-induced acute kidney injury

Acute kidney injury (AKI) disrupts energy metabolism. Targeting metabolism through AMP-activated protein kinase (AMPK) may alleviate AKI. ATX-304, a pan-AMPK activator, was evaluated in C57Bl/6 mice and tubular epithelial cell (TEC) cultures. Mice received ATX-304 (1 mg/g) or control chow for 7 days before cisplatin-induced AKI (CI-AKI). Primary cultures of tubular epithelial cells (TECs) were pre-treated with ATX-304 (20 µM, 4 h) prior to exposure to cisplatin (20 µM, 23 h). ATX-304 increased acetyl-CoA carboxylase phosphorylation, indicating AMPK activation. It protected against CI-AKI measured by serum creatinine (control 0.05 + 0.03 mM vs ATX-304 0.02 + 0.01 mM, P = 0.03), western blot for neutrophil gelatinase-associated lipocalin (NGAL) (control 3.3 + 1.8-fold vs ATX-304 1.2 + 0.55-fold, P = 0.002), and histological injury (control 3.5 + 0.59 vs ATX-304 2.7 + 0.74, P = 0.03). In TECs, pre-treatment with ATX-304 protected against cisplatin-mediated injury, as measured by lactate dehydrogenase release, MTS cell viability, and cleaved caspase 3 expression. ATX-304 protection against cisplatin was lost in AMPK-null murine embryonic fibroblasts. Metabolomic analysis in TECs revealed that ATX-304 (20 µM, 4 h) altered 66/126 metabolites, including fatty acids, tricarboxylic acid cycle metabolites, and amino acids. Metabolic studies of live cells using the XFe96 Seahorse analyzer revealed that ATX-304 increased the basal TEC oxygen consumption rate by 38%, whereas maximal respiration was unchanged. Thus, ATX-304 protects against cisplatin-mediated kidney injury via AMPK-dependent metabolic reprogramming, revealing a promising therapeutic strategy for AKI.

ApTOLL: A new therapeutic aptamer for cytoprotection and (re)myelination after multiple sclerosis.

ApTOLL is an aptamer selected to antagonize toll-like receptor 4 (TLR4), a relevant actor for innate immunity involved in inflammatory responses in multiple sclerosis (MS) and other diseases. The currently available therapeutic arsenal to treat MS is composed of immunomodulators but, to date, there are no (re)myelinating drugs available in clinics. In our present study, we studied the effect of ApTOLL on different animal models of MS. The experimental autoimmune encephalomyelitis (EAE) model was used to evaluate the effect of ApTOLL on reducing the inflammatory component. A more direct effect on oligodendroglia was studied with the cuprizone model and purified primary cultures of murine and human oligodendrocyte precursor cells (OPCs) isolated through magnetic-activated cell sorting (MACS) from samples of brain cortex. Also, we tested these effects in an ex vivo model of organotypic cultures demyelinated with lysolecithin (LPC). ApTOLL treatment positively impacted the clinical symptomatology of mice in the EAE and cuprizone models, which was associated with better preservation plus restoration of myelin and oligodendrocytes in the demyelinated lesions of animals. Restoration was corroborated on purified cultures of rodent and human OPCs. Our findings reveal a new therapeutic approach for the treatment of inflammatory and demyelinating diseases such as MS. The molecular nature of the aptamer exerts not only an anti-inflammatory effect but also neuroprotective and remyelinating effects. The excellent safety profile demonstrated by ApTOLL in animals and humans opens the door to future clinical trials in MS patients.

DsRNAi-mediated silencing of PIAS2beta specifically kills anaplastic carcinomas by mitotic catastrophe

The E3 SUMO ligase PIAS2 is expressed at high levels in differentiated papillary thyroid carcinomas but at low levels in anaplastic thyroid carcinomas (ATC), an undifferentiated cancer with high mortality. We show here that depletion of the PIAS2 beta isoform with a transcribed double-stranded RNA–directed RNA interference (PIAS2b-dsRNAi) specifically inhibits growth of ATC cell lines and patient primary cultures in vitro and of orthotopic patient-derived xenografts (oPDX) in vivo. Critically, PIAS2b-dsRNAi does not affect growth of normal or non-anaplastic thyroid tumor cultures (differentiated carcinoma, benign lesions) or cell lines. PIAS2b-dsRNAi also has an anti-cancer effect on other anaplastic human cancers (pancreas, lung, and gastric). Mechanistically, PIAS2b is required for proper mitotic spindle and centrosome assembly, and it is a dosage-sensitive protein in ATC. PIAS2b depletion promotes mitotic catastrophe at prophase. High-throughput proteomics reveals the proteasome (PSMC5) and spindle cytoskeleton (TUBB3) to be direct targets of PIAS2b SUMOylation at mitotic initiation. These results identify PIAS2b-dsRNAi as a promising therapy for ATC and other aggressive anaplastic carcinomas.

Exploring Ca2+ Dynamics in Myelinating Oligodendrocytes through rAAV-Mediated jGCaMP8s Expression in Developing Spinal Cord Organ Cultures.

Oligodendrocytes, the myelin-producing glial cells of the central nervous system (CNS), crucially contribute to myelination and circuit function. An increasing amount of evidence suggests that intracellular calcium (Ca2+) dynamics in oligodendrocytes mediates activity-dependent and activity-independent myelination. Unraveling how myelinating oligodendrocytes orchestrate and integrate Ca2+ signals, particularly in relation to axonal firing, is crucial for gaining insights into their role in the CNS development and function, both in health and disease. In this framework, we used the recombinant adeno-associated virus/Olig001 capsid variant to express the genetically encoded Ca2+ indicator jGCaMP8s, under the control of the myelin basic protein promoter. In our study, this tool exhibits excellent tropism and selectivity for myelinating and mature oligodendrocytes, and it allows monitoring Ca2+ activity in myelin-forming cells, both in isolated primary cultures and organotypic spinal cord explants. By live imaging of myelin Ca2+ events in oligodendrocytes within organ cultures, we observed a rapid decline in the amplitude and duration of Ca2+ events across different in vitro developmental stages. Active myelin sheath remodeling and growth are modulated at the level of myelin-axon interface through Ca2+ signaling, and, during early myelination in organ cultures, this phase is finely tuned by the firing of axon action potentials. In the later stages of myelination, Ca2+ events in mature oligodendrocytes no longer display such a modulation, underscoring the involvement of complex Ca2+ signaling in CNS myelination.