Large scale recovery and characterization of stromal cell-associated primitive haemopoietic progenitor cells from filter-retained human bone marrow

Abstract

Bone marrow aspirates are composed of two cellular compartments, an abundant buffy coat suspension and a minor particulate fraction. The particulate fraction is routinely removed by filtration prior to transplantation in order to reduce the risk of embolism. This study shows that the filter-retained fraction includes many multicellular complexes, previously defined as haematons. A haematon is a finely arborized stromal-web which is tightly packed with haemopoietic progenitor cells and differentiated postmitotic cells. Comparison of the pooled buffy coat and the filter-retained materials from healthy donors showed that the haematon fraction contained 8–40 × 10⁶ CD34⁺ cells, 20–115 × 10³ high proliferative potential colony-forming cells (HPP-CFC) and 0.49–2.67 × 10⁶ granulocyte–macrophage colony-forming unit (GM-CFU) which constituted 24 ± 8% (10–36; n = 8) of the total GM-CFU population harvested. Similar, but more variable recoveries of GM-CFU were obtained from the haematon fractions from patients with breast cancer (21 ± 13%; n = 10), Hodgkin’s disease (33 ± 19%; n = 4), non-Hodgkin’s lymphoma (21 ± 18; n = 7), but the recovery was lower from patients with acute myelogenous leukaemia (AML) (13 ± 13%; n = 6). The haematon fraction was enriched in CD34⁺ cells (2.5-fold), long-term culture initiating cells (LTC-IC/CAFC, week 5) (3.5-fold), HPP-CFC (2.8-fold) and GM-CFU (2.3-fold) over the buffy coat. Purified CD34⁺ cells expanded exponentially and produced 800 to 4000-fold more nucleated cells, 300 to 3500-fold more GM-CFU and 10 to 80-fold more HPP-CFC in stroma-free suspension culture with interleukin-1 (IL-1β), IL-3, IL-6, GM-CSF and stem cell factor (SCF), than did the starting cell input. The haematon fraction produced significantly more progenitor cells than the buffy coat in long-term liquid culture (LTC). This was due to the higher frequency of LTC-IC/CAFC and to the presence of the whole spectrum of native, stroma cell-associated CAFC in haematons. Thus, the haematon includes the most productive haematogenous compartment in human BM. This simple enrichment strategy, using filter-retained haematons, provides a rational source of BM cells for large scale experimental and/or clinical studies on haemopoietic stem cells and on critical accessory stromal cells.