Stachura, D. L. | bioRxiv Science

bioRxiv2025

Validation of Extracellular Vesicles in FACTORFIVEs EXOFECTA LyoBeads

ObjectiveThe objective of this project was to determine if extracellular vesicles (EVs) were present in FACTORFIVEs EXOFECTA LyoBeads to validate flash freezing and subsequent lyophilization as an effective EV preservation method. SummaryEVs were isolated from resuspended EXOFECTA LyoBeads using differential ultracentrifugation and subjected to further characterization techniques such as nanoparticle tracking analysis (NTA), single particle flow cytometry, and cryo-electron microscopy (cryo-EM). First, NTA revealed particles with size distributions and concentrations consistent with the expected characteristics of EVs, indicating successful isolation from samples. Secondly, flow cytometry was performed using antibodies targeting the canonical EV tetraspanins CD9, CD63, and CD81. Detection of these markers supported the presence of enriched EVs in the samples. Finally, cryo-EM was performed to confirm the existence of vesicular bodies and their morphology. Visualization of the samples revealed membrane-enclosed particles with a visible lipid bilayer, consistent with EVs. Overall, the results support EVs were in EXOFECTA LyoBeads, indicating EV retention in the final product after FACTORFIVEs method of flash freezing, stabilizing, and lyophilization.

molecular biology↗

bioRxiv2025

rapunzel5 is necessary for normal hematopoietic development in zebrafish

The molecular mechanisms regulating the highly complex process of hematopoiesis in vertebrates is still enigmatic. This system begins with the controlled differentiation of adult hematopoietic stem and progenitor cells (HSPCs), which can proliferate and generate all types of mature blood cells. Identifying the underlying factors and mechanisms that allow HSPCs to differentiate and proliferate is an essential issue in stem cell biology and tissue homeostasis; disruptions in these processes can cause severe diseases. A transcriptomic screen of hematopoietic-supportive zebrafish stromal tissues identified rapunzel5 (rpz5) was likely involved in vertebrate hematopoiesis. We performed loss-of-function experiments in zebrafish embryos at the one-cell-stage with morpholinos to determine if blood cell production was affected by rpz5. rpz5 knockdown resulted in reduced amounts of red blood cells, myeloid cells, and thrombocytes, and adding back exogenous rpz5 rescued these deficiencies. Further analysis with methylcellulose assays indicated that there was also a significant reduction in HSPCs following rpz5 reduction. Together, these findings suggest that zebrafish rpz5 is essential for normal formation, differentiation, and proliferation of HSPCs, specifically down the erythroid and myeloid pathway. Fully understanding the roles of novel genes such as rpz5 is essential for understanding the evolution of vertebrate hematopoiesis and for treating hematological diseases in the future.

genetics↗