#OA1-AM25-MN-18 - Ex Vivo and In Vivo Potency of CD34+ Hematopoietic Stem and Progenitor Cells Isolated from Cord Blood Using a Novel Technology

Immunomagnetic enrichment of CD34+ hematopoietic stem/progenitor cells from umbilical cord blood is an essential tool to generate humanized immune systems in immunodeficient (NSG) mice for research. Commercially available column-based kits are widely used for CD34+ isolation in both research and clinical applications. FerroBio is a newly developed, semi-automated CD34+ isolation technology, and an alternative to current column-based systems. This study was designed to directly examine the ex vivo and in vivo potency of FerroBio CD34+ cell isolates compared to cells isolated with a current commercially available column-based kit.

Study

Design/Methods: Fresh cord blood units (n=3) were split evenly by volume and CD34+ cells were isolated from each half with either FerroBio or Miltenyi Ultrapure Microbead kit. Pre- and post-thaw cells were characterized by immunophenotyping with flow cytometry. Ex vivo functional performance was assessed using colony-forming unit (CFU) and proliferation assays. In vivo, hematopoietic cell potency was assessed in NSG mice (n=9 per isolation group). Mice were sublethally irradiated and injected via tail vein with either 1E+04, 2.5E+03, or 0.5E+02 cells and peripheral blood was collected every 4 weeks to measure human chimerism and engraftment frequency. At 16 weeks, bone marrow will be collected, and repopulating cell frequency will be calculated. Secondary transplants of bone marrow into NSG mice are underway.

Results/Findings: Total pre-freeze purity (CD34+/total nucleated cells) was significantly higher in FerroBio isolates compared to control (79% vs 64%, respectively, t-test p < 0.05), while gated purity, viability, and CD34+ cell yields were similar. Post-thaw, both isolation techniques showed similar CFU and expansion capacities. Immunophenotyping revealed significantly higher numbers of primitive hematopoietic stem cells in FerroBio isolates compared to control (t-test, p < 0.05). Human chimerism in mouse peripheral blood following transplantation (% huCD45+) was significantly higher in FerroBio isolates in the 1E+04 dose (2-way ANOVA, p = 0.001) and the 2.5E+03 dose (p = 0.029) after 12 weeks (Figure 1).

Conclusions: FerroBio cell isolates have higher starting purity than cells isolated with the control, suggesting that column-based methods may co-isolate non-target cells. Although ex vivo performance was similar between both cell isolation methods, the FerroBio isolates produced significantly higher human chimerism, and mice trended toward faster engraftment in the highest dose. These results suggest that the CD34+ cell isolation technique may influence cell potency in vivo. These results also suggest that FerroBio offers a suitable and superior alternative to current column-based techniques that may be desirable in manufacturing or laboratory applications.