Large-Scale Selection of CD34+ Peripheral Blood Progenitors and Expansion of Neutrophil Precursors for Clinical Applications

Abstract

Hematopoietic recovery after high-dose chemotherapy is characterized by an obligate period of neutropenia of approximately 8–10 days. It is postulated that if a pool of neutrophil precursors and progenitors were expanded in vitro and reinfused, the duration of neutropenia may be substantially shortened by these cells capable of providing mature neutrophils within days of reinfusion. In this study, peripheral blood progenitor cell products were obtained from six normal donors mobilized with rhG-CSF and two patients mobilized with cyclophosphamide and rhG-CSF. CD34+ cells were isolated using the Isolex® immunomagnetic bead method. A mean of 8.26 × 10⁷ CD34+ cells with a mean purity of 74.5% were seeded at a concentration of 1 × 10⁵/ml into a 12 day stroma-free liquid culture using gas-permeable bags. A serum-free growth medium supplemented with PIXY321 was used. On day 7, there was a mean cellular expansion of fourfold, at which time the cells were resuspended at the initial concentration, yielding a mean culture volume of 3L (1–6 L). On day 12, there was an additional mean fold cellular expansion of 10 ×, achieving an overall mean fold expansion of 41 ± 16. Cellular characterization of the expanded cells revealed predominantly neutrophil precursors by morphology (mean 70.1%) and flow cytometric analysis. A mean of 52.3% of the expanded cells expressed CD15. Immunohistochemical staining revealed a mean of 7.1% CD41a+ megakaryocytic progenitors in the final cultured cell product. Detectable CD34+ cells were maintained only in those cultures initiated with greater than 90% CD34+ cells. Colony-forming units-granulocyte-macrophage (CFU-GM) were maintained in the 12 day culture at a level similar to the preculture number, whereas CFU mixed were depleted in all samples. On day 0, there were few CFU clusters (colonies containing fewer than 50 cells) identified, but by day 12, a mean total of 8.3 × 10⁶ CFU clusters were identified. On day 12, the expanded cells were harvested and pooled using the Fenwal CS3000® Plus blood cell separator and resuspended in Plasma-Lyte®-A with 1% human serum albumin. The mean harvest recovery of expanded progenitors was 91%, with a mean viability of 86%.