Simple 3D spheroid cell culture model for studies of prion infection.

Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 µm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, ß-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.

Large extracellular vesicles transfer more prions and infect cell culture better than small extracellular vesicles.

Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in ß-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of ß-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small EVs.

Flow Cytometry Analysis of Blood Large Extracellular Vesicles in Patients with Multiple Sclerosis Experiencing Relapse of the Disease.

The number of people living with multiple sclerosis (MS) in developed countries is increasing. The management of patients is hindered by the absence of reliable laboratory tests accurately reflecting the disease activity. Extracellular vesicles (EVs) of different cell origin were reportedly elevated in MS patients. We assessed the diagnostic potential, with flow cytometry analysis, of fresh large EVs (lEVs), which scattered more light than the 590 nm silica beads and were isolated from the blood plasma of relapsing remitting MS patients. Venous blood was collected from 15 patients and 16 healthy controls (HC). The lEVs were isolated from fresh platelet-free plasma by centrifugation, labelled with antibodies and the presence of platelet (CD41+, CD36+), endothelial (CD105+), erythrocyte (CD235a+), leukocyte (CD45+, CD19+, CD3+) and phosphatidylserine (Annexin V+) positive lEVs was analyzed using standard flow cytometry. Cryo-electron microscopy was used to verify the presence of EVs in the analyzed plasma fractions. MS patients experiencing acute relapse had slightly reduced relative levels (% of positive lEVs) of CD105+, CD45+, CD3+, CD45+CD3+ or CD19+ labelled lEVs in comparison to healthy controls. An analysis of other markers or a comparison of absolute lEV counts (count of lEVs/µL) did not yield any significant differences. Our data do not support the hypothesis that the exacerbation of the disease in RRMS patients leads to an increased numbers of circulating plasma lEVs which can be monitored by standard flow cytometry.

Large Platelet and Endothelial Extracellular Vesicles in Cord Blood of Preterm Newborns: Correlation with the Presence of Hemolysis.

Different biomarkers are investigated to detect the causes of severe complications in preterm infants. Extracellular vesicles (EVs) are recognized as an important part of cell-to-cell communication, and their increased levels were reported in numerous pathological states. We aimed to increase our knowledge about the incidence of platelet and endothelial EVs in cord blood of preterm newborns using conventional flow cytometry. The presence of platelet (CD36+CD41+), activated platelet (CD41+CD62+), and endothelial (CD31+CD105+) EVs was analyzed. Immune electron microscopy was used to confirm the presence of EVs and the specificity of their labeling. The size of detected extracellular vesicles was in the range 400-2000 nm. The differences in the counts of EVs between the preterm and control group were not significant and no correlation of EVs count with gestation age was recorded. Cord blood plasma samples with free hemoglobin level > 1 mg/mL had more than threefold higher counts of CD36+CD41+ and CD41+CD62+ EVs (p < 0.001), while the count of CD31+CD105+ EVs was only moderately increased (p < 0.05). Further studies utilizing cytometers with improved sensitivity are needed to confirm that the analysis of large platelet and endothelial EVs mirrors the quantitative situation of their whole plasma assemblage.

Cord Blood Extracellular Vesicles Analyzed by Flow Cytometry with Thresholding Using 405 nm or 488 nm Laser Leads to Concurrent Results.

Extracellular vesicles (EVs) from liquid biopsies are extensively analyzed by flow cytometry, a technology that is continuously evolving. Thresholding utilizing a violet 405 nm laser side scatter (VSSC) has recently been implemented. Here, we collected set of large EV (lEV) samples from cord blood, which we analyzed using a standard flow cytometer improved via a 405 nm laser side scatter. Samples were analyzed using two distinct thresholding methods-one based on VSSC, and one based on VSSC combined with fluorescence thresholding on stained phosphatidylserine. Through these thresholding methods, we compared lEVs from pre-term births and control cord blood. Double-labeled lEVs with platelet CD36+/CD41+, activated platelet CD41+/CD62P+ and endothelial CD31+/CD105+ antibodies were used. Apart from comparing the two groups together, we also correlated measured lEVs with the thresholding methods. We also correlated the results of this study with data analyzed in our previous study in which we used a conventional 488 nm laser SSC. We did not find any difference between the two cord blood groups. However, we found highly concurrent data via our correlation of the thresholding methods, with correlation coefficients ranging from 0.80 to 0.96 even though the numbers of detected lEVs differed between thresholding methods. In conclusion, our approaches to thresholding provided concurrent data and it seems that improving the cytometer with the use of a VSSC increases its sensitivity, despite not being particularly critical to the validity of flow cytometric studies that compare pathological and physiological conditions in liquid biopsies.

Changes in cellular prion protein expression, processing and localisation during differentiation of the neuronal cell line CAD 5.

BACKGROUND INFORMATION: Cellular prion protein (PrPC ) is infamous for its role in prion diseases. The physiological function of PrPC remains enigmatic, but several studies point to its involvement in cell differentiation processes. To test this possibility, we monitored PrPC changes during the differentiation of prion-susceptible CAD 5 cells, and then we analysed the effect of PrPC ablation on the differentiation process. RESULTS: Neuronal CAD 5 cells differentiate within 5 days of serum withdrawal, with the majority of the cells developing long neurites. This process is accompanied by an up to sixfold increase in PrPC expression and enhanced N-terminal ß-cleavage of the protein, which suggests a role for the PrPC in the differentiation process. Moreover, the majority of PrPC in differentiated cells is inside the cell, and a large proportion of the protein does not associate with membrane lipid rafts. In contrast, PrPC in proliferating cells is found mostly on the cytoplasmic membrane and is predominantly associated with lipid rafts. To determine the importance of PrPC in cell differentiation, a CAD 5 PrP-/- cell line with ablated PrPC expression was created using the CRISPR/Cas9 system. We observed no considerable difference in morphology, proliferation rate or expression of molecular markers between CAD 5 and CAD 5 PrP-/- cells during the differentiation initiated by serum withdrawal. CONCLUSIONS: PrPC characteristics, such as cell localisation, level of expression and posttranslational modifications, change during CAD 5 cell differentiation, but PrPC ablation does not change the course of the differentiation process. SIGNIFICANCE: Ablation of PrPC expression does not affect CAD 5 cell differentiation, although we observed many intriguing changes in PrPC features during the process. Our study does not support the concept that PrPC is important for neuronal cell differentiation, at least in simple in vitro conditions.

The encapsidation of polyomavirus is not defined by a sequence-specific encapsidation signal.

Mouse polyomavirus (MPyV) is considered a potential tool for the application of gene therapy; however, the current knowledge of the encapsulation of DNA into virions is vague. We used a series of assays based on the encapsidation of a reporter vector into MPyV pseudovirions to identify putative cis-acting elements that are involved in DNA encapsidation. None of the sequences that were derived from MPyV have been shown to solely enhance the encapsidation of a reporter vector in the assay. The frequency of encapsidation strongly correlated with the total intracellular amount of the vector after transfection. The encapsidation of target DNA into the pseudovirions was shown to be non-specific, and the packaging of non-replicated DNA was observed. We propose that the actual concentration of target DNA at the sites of virion formation is the primary factor that determines its selection for encapsidation.