TRANSPLANTATION AND CELLULAR ENGINEERING
A novel method of CD34+ cell separation from umbilical cord blood
- Appropriate permission and informed consent of the cord blood donors were obtained by the consultant gynecologist for in vitro studies reported here.
Umbilical cord blood (UCB) is rich in the heavily glycosylated CD34 antigen–bearing hematopoietic stem cells that are valuable for transplantation therapy of malignant and nonmalignant disease. CD34+ cell yields (0.13%-0.25%-0.3%) of mononuclear cells (UCMCs) isolated by anti-CD34 monoclonal antibody (MoAb) on immunomagnetic particles (e.g., Miltenyi particles) are insufficient to treat adults.
Study Design and Methods
We fractionated UCMCs by physicochemical charge-based methods. Avoiding Miltenyi particles and HESPAN yielded 30 x 106 to 54 x 106 UCMCs/20 mL UCB by Ficoll-Isopaque for serial depletion fractionation, using nylon wool column (NWC) or direct rosetting with sheep red blood cells (SRBCs) without incubation in the cold.
CD34+ cell yields (approx. 5.12%) were 39 times greater than 0.13% (Korean study, 11,098 UCB units) and 10 to 20 times greater than 0.25% to 0.3% harvested by anti-CD34 Miltenyi particles. SRBC depletion of most high-specific-gravity T cells achieved considerable enrichment of CD34+ and BY55+ cells. Using NWC achieved 2.5-fold enrichment of CD34+ cells and twofold enrichment of BY55+ cells. Direct SRBC rosetting provided better or higher enrichment of CD34+ cells. Overall CD34+ cell yield in low-density fraction was more than twice after direct rosetting (38% vs. 16%) in contrast to separation by NWC followed by SRBC rosetting. CD3+ cell yields (by three CD markers) were approximately 8.83%, far below approximately 30 x 107/kg considered acceptable to avoid graft-versus-host disease. Natural killer cell yields (CD16+/CD56+ and BY55+/ CD160+) are in perfect agreement.
Achieving approximately 5% CD34+ cell yields from single UCB donations, a major advance, holds great promise for CD34+ cell therapy of adults and larger children, and cheaper cultured RBC manufacture.