The profile of circulating extracellular vesicles depending on the age of the donor potentially drives the rejuvenation or senescence fate of hematopoietic stem cells.

Blood donor age has become a major concern due to the age-associated variations in the content and concentration of circulating extracellular nano-sized vesicles (EVs), including exosomes. These EVs mirror the state of their parental cells and transfer it to the recipient cells via biological messengers such as microRNAs (miRNAs, miRs). Since the behavior of hematopoietic stem cells (HSCs) is potentially affected by the miRs of plasma-derived EVs, a better understanding of the content of EVs is important for the safety and efficacy perspectives in blood transfusion medicine. Herein, we investigated whether the plasma-derived EVs of young (18-25 years) and elderly human donors (45-60 years) can deliver "youth" or "aging" signals into human umbilical cord blood (hUCB)-derived HSCs in vitro. The results showed that EVs altered the growth functionality and differentiation of HSCs depending on the age of the donor from which they are derived. EVs of young donors could ameliorate the proliferation and self-renewal potential of HSCs whereas those of aged donors induced senescence-associated differentiation in the target cells, particularly toward the myeloid lineage. These findings were confirmed by flow cytometric analysis of surface markers and microarray profiling of genes related to stemness (e.g., SOX-1, Nanog) and differentiation (e.g., PU-1). The results displayed an up-regulation of miR-29 and miR-96 and a down-regulation of miR-146 in EVs derived from elderly donors. The higher expression of miR-29 and miR-96 contributed to the diminished expression of CDK-6 and CDKN1A (p21), promoting senescence fate via cell growth suppression, while the lower expression of miR-146 positively regulates TRAF-6 expression to accelerate biological aging. Our findings reveal that plasma-derived EVs from young donors can reverse the aging-associated changes in HSCs, while vice versa, the EVs from elderly donors rather promote the senescence process.

Long-term Effects of Regular Whole Blood Donation on Peripheral Blood CD34+ Cells Population with using Leukoreduction Filters.

Background & Objective: Trapped cell population in leukoreduction filters (LRFs) contains such a significant number of CD34+ hematopoietic stem cells that can be recovered to be used in research studies. Methods: Samples (n=20) were obtained from 10 first-time donors and 10 regular blood donors with more than 30 times blood donation. After separating leukocytes from LRFs by backflushing, total leukocyte number and differential count were determined in both groups using an automated haemocytometer. Then cell viability and CD34+ cell quantification were assessed using 7- amino-actinomycin D and fluorescent-labeled monoclonal antibodies using flow cytometry, respectively. Results: Total leukocyte count was 665±164.92×106 in the first-time blood donors and 883±233.89×106 in the regular donors, which were not significantly different (P=0.08). While the number of CD34+ cells was significantly reduced in the regular donors compared to the first-time donors (0.58±0.20×106/µL vs. 0.36±0.22×106/µL; P=0.034). There was no significant difference in terms of absolute neutrophil count (10.58±3.66×06 vs. 13.17±6.45×106/µL; P=0.349), lymphocytes (7.75±3.11×106 vs. 10.38±3.77×106 /µL; P=0.917), and monocytes (2.31±0.88×106 vs. 2.59±1.09×106/µL; P=0.591) between the first-time and regular donor groups, respectively. Based on the correlation coefficients, the participants' age had no significant effect on these variables. Conclusion: The results of this study depicted that regular blood donation reduces the number of CD34+ cells in the peripheral blood (PB) of regular donors while it has no significant effect on the ratio of myeloid to lymphoid cells of the two groups.

Optimizing the recovery of peripheral blood mononuclear cells trapped in leukoreduction filters - A comparison study

Abstract Introduction The isolation of captured peripheral blood mononuclear cells (PBMNCs) from leukoreduction filters (LRFs) can be of great importance in terms of bringing the lost cells back into use. Objective The aim of this study was to evaluate various methods based on their potential to recover the peripheral blood cells from LRFs with a focus on mononuclear cells (MNCs). Method For cell isolation from LRFs, three distinct methods (back-flushing, direct and vacuum pump) were compared through the calculation of the yield of isolated MNCs. The viability of extracted cells was determined by the flow cytometry technique. Moreover, the recovered MNCs were characterized regarding the presence of blood stem cell purification. The cell culture, microscopic observation, and immunophenotyping were employed to characterize the blood stem cells (hematopoietic, mesenchymal and progenitor endothelial stem cells). Results The yield of isolation obtained in the back-flushing, direct and vacuum pump methods were 17.7 ± 1.28, 17.3 ± 0.96 and 21.2 ± 0.90 percent, respectively. Although the highest potential for total blood cell recovery belonged to the vacuum pump method, the lowest cell viability (85.73 ± 4.84%) was observed in this method. However, the isolation process of the back-flushing and direct methods had less effect on cell viability. The characterization of the isolated MNCs displayed that the dominant positive phenotype was for CD34/CD45, indicating hematopoietic stem cells. In addition, the endothelial stem/progenitor cells were significantly detected as CD31/CD133 positive cells. Conclusion According to our results and considering the safety and efficiency potential of each of the applied methods, the back-flushing in comparison with the other methods can be considered a suitable procedure for MNC isolation from LRFs.

Optimizing the recovery of peripheral blood mononuclear cells trapped in leukoreduction filters - A comparison study.

INTRODUCTION: The isolation of captured peripheral blood mononuclear cells (PBMNCs) from leukoreduction filters (LRFs) can be of great importance in terms of bringing the lost cells back into use. OBJECTIVE: The aim of this study was to evaluate various methods based on their potential to recover the peripheral blood cells from LRFs with a focus on mononuclear cells (MNCs). METHOD: For cell isolation from LRFs, three distinct methods (back-flushing, direct and vacuum pump) were compared through the calculation of the yield of isolated MNCs. The viability of extracted cells was determined by the flow cytometry technique. Moreover, the recovered MNCs were characterized regarding the presence of blood stem cell purification. The cell culture, microscopic observation, and immunophenotyping were employed to characterize the blood stem cells (hematopoietic, mesenchymal and progenitor endothelial stem cells). RESULTS: The yield of isolation obtained in the back-flushing, direct and vacuum pump methods were 17.7 ±â€¯1.28, 17.3 ±â€¯0.96 and 21.2 ±â€¯0.90 percent, respectively. Although the highest potential for total blood cell recovery belonged to the vacuum pump method, the lowest cell viability (85.73 ±â€¯4.84%) was observed in this method. However, the isolation process of the back-flushing and direct methods had less effect on cell viability. The characterization of the isolated MNCs displayed that the dominant positive phenotype was for CD34/CD45, indicating hematopoietic stem cells. In addition, the endothelial stem/progenitor cells were significantly detected as CD31/CD133 positive cells. CONCLUSION: According to our results and considering the safety and efficiency potential of each of the applied methods, the back-flushing in comparison with the other methods can be considered a suitable procedure for MNC isolation from LRFs.