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Background: Cryopreservation and thawing protocols represent key factors for the efficacy of cellular therapy products, such as hematopoietic stem cells (HSCs). While the HSC cryopreservation has already been standardized, the thawing procedures have been poorly studied. This study aimed to evaluate the thawing and washing protocol of cord blood (CB) derived HSCs or the HPC(CB), by selecting the optimal thawing solution and determining CD34+ cells' stability over time., Study Design and Methods: Seven cryopreserved CB products were thawed, washed, and resuspended in three different solutions (10% Dextran40 in NaCl equally prepared with 5% human albumin; 5% human albumin in PBS/EDTA; and normal saline) and stored at 4°C (±2°C). Mononuclear cell (MNC) count, CD45+/CD34+ cell enumeration, and cell viability were tested at 0, 1, 2, 4, 6, 8, 12, 24, 36, and 48 h. The protocol with the selected solution was further validated on additional 10 CB samples. The above parameters and the colony-forming unit (CFU) assay were analyzed at time points 0, 2, 4, 6, and 8 h., Results and Discussion: The results showed that the 5% human albumin was the most suitable thawing solution. MNCs were stable up to 4 h (p = 0.009), viable CD45+ cells were unstable even at 2 h (p = 0.013), and viable CD34+ cells were stable until 6 h (p = 0.019). The CFU assay proved the proliferative potential up to 8 h, although significantly decreased after 4 h (p = 0.013), and correlated with the viable CD34+ cell counts. We demonstrated that the post-thawed and washed HPC(CB) using 5% human albumin is stable for up to 4 h., (© 2023 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Emerging gene therapy clinical trials test the correction of hemophilia A (HA) by replacing factor VIII (FVIII) in autologous hematopoietic stem cells (HSCs). Although it is known that platelets, monocyte/macrophages, and mesenchymal stromal cells can secrete transgenic FVIII, a systematic examination of blood lineages as extrahepatic sources of FVIII, to our knowledge, has not yet been performed. In this study, we sought to provide a comprehensive map of native and lentivirus-based transgenic FVIII production from HSC stage to mature blood cells, through a flow cytometry analysis. In addition, we generated a model of transient HA in zebrafish based on antisense RNA, to assess the corrective potential of the FVIII-transduced HSCs. We discovered that FVIII production begins at the CD34+ progenitor stage after cytokine stimulation in culture. Among all mature white blood cells, monocytes are the largest producers of native FVIII and can maintain protein overexpression during differentiation from HSCs when transduced by a FVIII lentiviral vector. Moreover, the addition of the HSC self-renewal agonist UM171 to CD34+ cells during transduction expanded a subpopulation of CD14+/CD31+ monocytes with excellent ability to carry the FVIII transgene, allowing the correction of HA phenotype in zebrafish. Finally, the HA zebrafish model showed that f8 RNA is predominantly localized in the hematopoietic system at the larval stage, which indicates a potential contributory role of FVIII in hematopoiesis that warrants further investigation. We believe that this study may be of broad interest to hematologists and researchers striving to advance knowledge and permanent treatments for patients with HA., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)