365 - Enabling efficient ex vivo expansion of long-term human hematopoietic stem and progenitor cells for cell therapy

Abstract

Umbilical-cord blood (UCB) is an important source of hematopoietic stem and progenitor cells (HSPC) for transplantation into patients lacking a suitable HLA-matched donor. However, due to the limited cell dose in each cord blood unit, individuals >60 kg are restricted from the use of UCB-based therapy. Ex vivo expansion of UCB CD34+ cells is one strategy employed to increase the hematopoietic cell dosage. A major limitation of current systems used for the expansion of HSPC is that ex vivo culture leads to expansion and differentiation at the expense of the most primitive pluripotent long-term stem cells. This has limited the clinical application of ex vivo expanded HSPC, since short-term progenitor cells only provide transient protection, ultimately reducing the long-term positive health outcomes, increasing the duration of hospitalizations, and health care costs per patient. Development of a culture system that expands both short-term and long-term HSPC would facilitate immune protection during the early phase of recovery, and provides a suitable solution for transfusion-independent hematopoiesis. Therefore, we sought to develop a HSPC culture medium that enables the expansion of both long-term and short-term HSPC, while maintaining their functional properties. To this end, we conducted several iterative rounds of Design of Experiments (DOE) involving multifactorial analysis, and mathematical modeling methods. Definitive Screening DOEs allowed us to identify optimal combinations and concentrations of essential media components, small molecules, and growth factors. The performance of candidate HSPC expansion media were evaluated after 7 days of culture, with the following attributes assessed: (1) viability of cells; (2) numbers of total nucleated cells; (3) percentages and numbers of CD34+ cells; (4) percentages and numbers of CD34+CD90+CD45RA- cells; (5) expression of aldehyde dehydrogenase by expanded CD34+ cells; and (6) colony-forming unit (CFU) assays. As the transplantation of HSPC in immuno-deficient mice is the gold standard in determining whether the expanded cells are engraftable, we plan to conduct these studies with the lead candidate HSPC expansion medium. Taken together, we seek to highlight our design philosophy in HSPC culture media development. We believe that our efforts are critical for the successful utilization of hematopoietic stem cell transplants in translational cell therapies.