The phenotype of cryopreserved platelets influences the formation of platelet-leukocyte aggregates in an in vitro model.

Cryopreservation significantly alters the phenotype of platelets; generating distinct subpopulations, which may influence the formation of platelet leukocyte aggregates (PLA). PLAs are immunomodulatory and have been associated with transfusion-associated adverse events. As such, the aim of this study was to examine the effect of cryopreservation on the ability of platelets to form PLAs, using a monocyte-like cell line (THP-1). Platelets were tested pre-freeze, post-thaw and following stimulation with TRAP-6 or A23187, both alone and following co-culture with THP-1 cells for 1 and 24 hours (n = 6). Platelet subpopulations and platelet-THP-1 cell aggregates were analyzed using multi-color imaging flow cytometry using Apotracker Green (ApoT), CD42b, CD62P, CD61, and CD45. Cryopreservation resulted in the generation of activated (ApoT-/CD42b+/CD62P+), procoagulant (ApoT+/CD42b+/CD62P+) and a novel (ApoT+/CD42b+/CD62P-) platelet subpopulation. Co-incubation of cryopreserved platelets with THP-1 cells increased PLA formation compared to pre-freeze but not TRAP-6 or A23187 stimulated platelets. P-selectin on the surface membrane was correlated with increased PLA formation. Our findings demonstrate that cryopreservation increases the interaction between platelets and THP-1 cells, largely due to an increase in procoagulant platelets. Further investigation is required to determine the immunological consequences of this interaction.

What do we know? Cryopreserved platelets are an alternative to overcome issues with the short shelf-life of room-temperature stored plateletsAfter thawing, cryopreserved platelets exhibit changes in cell structure and receptor abundanceActivated platelets can attach to leukocytes, forming platelet-leukocyte aggregates and altering their immune functionPlatelet-leukocyte aggregates can increase inflammation, which is associated with adverse events after transfusion, which can negatively affect patient outcomesWhat did we discover? Cryopreservation results in a heterogenous mix of platelet subpopulationsCryopreserved platelets display increased adherence to a monocyte-like cell line (THP-1 cells). Platelet-THP-1 aggregate formation was linked to expression of CD62P on the surface of the plateletsThe increase in cryopreserved platelet-THP-1 cell aggregates was largely due to an increase in procoagulant plateletsWhat is the impact? Our data demonstrate that cryopreservation increases platelet interaction with a monocyte-like cell lineThis may mediate immune responses and/or circulation time of transfused platelets.

Adipose-Derived Stem Cells Spontaneously Express Neural Markers When Grown in a PEG-Based 3D Matrix.

Neurological diseases are among the leading causes of disability and death worldwide and remain difficult to treat. Tissue engineering offers avenues to test potential treatments; however, the development of biologically accurate models of brain tissues remains challenging. Given their neurogenic potential and availability, adipose-derived stem cells (ADSCs) are of interest for creating neural models. While progress has been made in differentiating ADSCs into neural cells, their differentiation in 3D environments, which are more representative of the in vivo physiological conditions of the nervous system, is crucial. This can be achieved by modulating the 3D matrix composition and stiffness. Human ADSCs were cultured for 14 days in a 1.1 kPa polyethylene glycol-based 3D hydrogel matrix to assess effects on cell morphology, cell viability, proteome changes and spontaneous neural differentiation. Results showed that cells continued to proliferate over the 14-day period and presented a different morphology to 2D cultures, with the cells elongating and aligning with one another. The proteome analysis revealed 439 proteins changed in abundance by >1.5 fold. Cyclic nucleotide 3'-phosphodiesterase (CNPase) markers were identified using immunocytochemistry and confirmed with proteomics. Findings indicate that ADSCs spontaneously increase neural marker expression when grown in an environment with similar mechanical properties to the central nervous system.

Neural Marker Expression in Adipose-Derived Stem Cells Grown in PEG-Based 3D Matrix Is Enhanced in the Presence of B27 and CultureOne Supplements.

Adipose-derived stem cells (ADSCs) have incredible potential as an avenue to better understand and treat neurological disorders. While they have been successfully differentiated into neural stem cells and neurons, most such protocols involve 2D environments, which are not representative of in vivo physiology. In this study, human ADSCs were cultured in 1.1 kPa polyethylene-glycol 3D hydrogels for 10 days with B27, CultureOne (C1), and N2 neural supplements to examine the neural differentiation potential of ADSCs using both chemical and mechanical cues. Following treatment, cell viability, proliferation, morphology, and proteome changes were assessed. Results showed that cell viability was maintained during treatments, and while cells continued to proliferate over time, proliferation slowed down. Morphological changes between 3D untreated cells and treated cells were not observed. However, they were observed among 2D treatments, which exhibited cellular elongation and co-alignment. Proteome analysis showed changes consistent with early neural differentiation for B27 and C1 but not N2. No significant changes were detected using immunocytochemistry, potentially indicating a greater differentiation period was required. In conclusion, treatment of 3D-cultured ADSCs in PEG-based hydrogels with B27 and C1 further enhances neural marker expression, however, this was not observed using supplementation with N2.

Comparisons of Protein and Peptide Complexity in Poneroid and Formicoid Ant Venoms.

Animal venom peptides are currently being developed as novel drugs and bioinsecticides. Because ants use venoms for defense and predation, venomous ants represent an untapped source of potential bioactive toxins. This study compared the protein and peptide components of the poneroid ants Neoponera commutata, Neoponera apicalis, and Odontomachus hastatus and the formicoid ants Ectatomma tuberculatum, Ectatomma brunneum, and Myrmecia gulosa. 1D and 2D PAGE revealed venom proteins in the mass range <10 to >250 kDa. NanoLC-ESI-QTOF MS/MS analysis of tryptic peptides revealed the presence of common venom proteins and also many undescribed proteins. RP-HPLC separation followed by MALDI-TOF MS of the venom peptides also revealed considerable heterogeneity. It was found that the venoms contained between 144 and 1032 peptides with 5-95% of peptides in the ranges 1-4 and 1-8 kDa for poneroid and formicoid ants, respectively. By employing the reducing MALDI matrix 1,5-diaminonapthalene, up to 28 disulfide-bonded peptides were also identified in each of the venoms. In particular, the mass range of peptides from poneroid ants is lower than peptides from other venoms, indicating possible novel structures and pharmacologies. These results indicate that ant venoms represent an enormous, untapped source of novel therapeutic and bioinsecticide leads.