Current Understanding of the Relationship between Blood Donor Variability and Blood Component Quality.

While differences among donors has long challenged meeting quality standards for the production of blood components for transfusion, only recently has the molecular basis for many of these differences become understood. This review article will examine our current understanding of the molecular differences that impact the quality of red blood cells (RBC), platelets, and plasma components. Factors affecting RBC quality include cytoskeletal elements and membrane proteins associated with the oxidative response as well as known enzyme polymorphisms and hemoglobin variants. Donor age and health status may also be important. Platelet quality is impacted by variables that are less well understood, but that include platelet storage sensitive metabolic parameters, responsiveness to agonists accumulating in storage containers and factors affecting the maintenance of pH. An increased understanding of these variables can be used to improve the quality of blood components for transfusion by using donor management algorithms based on a donors individual molecular and genetic profile.

Mesenchymal stem cells as a feeder layer can prevent apoptosis of expanded hematopoietic stem cells derived from cord blood.

Umbilical cord blood (UCB) has been used for transplantation in the treatment of hematologic disorders as a source of hematopoietic stem cells (HSCs). Because of insufficient number of cord blood CD34(+) cells, the expansion of these cells seems to be important for clinical application. Mesenchymal stromal cells (MSCs), playing an important role in HSCs maintenance, were used as feeder layer. Apoptosis and cell cycle distribution of expanded cells were analyzed in MSCs co-culture and cytokine conditions and results were compared. Three culture conditions of cord blood HSCs were prepared ex-vivo for 14 days: cytokines (SCF, TPO and Flt3L) with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs without cytokines. Expansion was followed by measuring the total nucleated cells (TNCs), CD34(+) ( ) cells and colony-forming unit (CFU) output. Flow cytometry analysis of stained cells by annexin V and propidium iodide was performed for detection of apoptosis rate and cell cycle distribution in expanded cells. Maximum cord blood CD34(+) cells expansion was observed in day 10. The mean fold change of TNCs and CD34+ cells at day 10 in the co-culture system with cytokines was significantly higher than the cytokine culture without MSCs feeder layer and co-culture system without cytokines (n=6, p=0.023). The highest apoptosis rate and the least number of cells in Go/G1 phase were observed in cytokine culture without feeder layer (p=0.041). The expansion of cord blood HSCs on MSCs as a feeder layer resulted in higher proliferation and reduction in apoptosis rate.

Expression of P16 cell cycle inhibitor in human cord blood CD34+ expanded cells following co-culture with bone marrow-derived mesenchymal stem cells.

BACKGROUND: Because of insufficient number of cord blood hematopoietic stem cells (CB-HSC), expansion of these cells seems to be important for clinical application in adults. Cell cycle inhibitors are important regulators in normal hematopoietic regeneration. In this study, mRNA expression and promoter methylation status of p16 were evaluated during CB-HSC ex vivo expansion using cytokines and a co-culture system with mesenchymal stem cells (MSCs) feeder layer. METHODS: ex vivo cultures of CB-HSCs were performed in three culture conditions for 14 days: cytokines with MSCs feeder layer, cytokines without MSCs feeder layer, and co-culture with MSCs without cytokine. After expansion, measuring total number of cells, CD34+ cells and colony-forming unit (CFU) assay was performed. Methylation status of the p16(INK4a) gene promoter was analyzed using methylation-specific polymerase chain reaction (PCR), and p16 mRNA expression was evaluated by real-time reverse transcriptase-PCR. RESULTS: Maximum CB-HSC expansion was observed in day 10 of expansion. The data showed that after 10 days, p16 mRNA expression in the expanded cells at the co-culture system without cytokine was higher than in CD34+ fresh cells (P < 0.01); however, p16 mRNA expression in the expanded cells at both cytokine cultures with and without MSCs feeder layer was decreased. p16 gene promoter of expanded CD34+ cells remained in unmethylated form just like fresh CD34+ cells in all the three culture conditions at days 5, 10, and 14 of culture. CONCLUSION: Expression in HSCs of p16(INK4a), an important cell cycle regulator in normal hematopoietic regeneration disruption of which is involved in leukemic cell development, was increased during 10 days of expansion in co-culture with MSCs feeder layers. Also, no methylation of p16 promoter was observed, which is capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.