A modified CD34+ hematopoietic stem and progenitor cell isolation strategy from cryopreserved human umbilical cord blood.
Mata MF., Hernandez D., Rologi E., Grandolfo D., Hassan E., Hua P., Kallmeier R., Hirani S., Heuts F., Tittrea V., Choo Y., Baradez M-O., Watt SM., Tarunina M.
BACKGROUND: Umbilical cord blood (UCB) is a source of hematopoietic stem cells for transplantation, offering an alternative for patients unable to find a matched adult donor. UCB is also a versatile source of hematopoietic stem and progenitor cells (hCD34 + HSPCs) for research into hematologic diseases, in vitro expansion, ex vivo gene therapy, and adoptive immunotherapy. For these studies, there is a need to isolate hCD34 + HSPCs from cryopreserved units, and protocols developed for isolation from fresh cord blood are unsuitable. STUDY DESIGN: This study describes a modified method for isolating hCD34 + HSPCs from cryopreserved UCB. It uses the Plasmatherm system for thawing, followed by CD34 microbead magnetic-activated cell sorting isolation with a cell separation kit (Whole Blood Columns, Miltenyi Biotec). hCD34 + HSPC phenotypes and functionality were assessed in vitro and hematologic reconstitution determined in vivo in immunodeficient mice. RESULTS: Total nucleated cell recovery after thawing and washing was 44.7 ± 11.7%. Recovery of hCD34 + HSPCs after application of thawed cells to Whole Blood Columns was 77.5 ± 22.6%. When assessed in two independent laboratories, the hCD34+ cell purities were 71.7 ± 10.7% and 87.8 ± 2.4%. Transplantation of the enriched hCD34 + HSPCs into NSG mice revealed the presence of repopulating hematopoietic stem cells (estimated frequency of 0.07%) and multilineage engraftment. CONCLUSION: This provides a simplified protocol for isolating high-purity human CD34 + HSPCs from banked UCB adaptable to current Good Manufacturing Practice. This protocol reduces the number of steps and associated risks and thus total production costs. Importantly, the isolated CD34 + HSPCs possess in vivo repopulating activity in immunodeficient mice, making them a suitable starting population for ex vivo culture and gene editing.