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

Bone marrow aspirates are composed of two cellular compartments, an abundan t huffy coat suspension and a minor particulate fraction. The particulate f raction is routinely removed by filtration prior to transplantation in orde r to reduce the risk of embolism. This study shows that the filter-retained fraction includes many multicellular complexes, previously defined as haem atons, A haematon is a finely arborized stromal-web which is tightly packed with haemopoietic progenitor cells and differentiated postmitotic cells. C omparison of the pooled buffy coat and the filter-retained materials from h ealthy donors showed that the haematon fraction contained 8-40 x 10(6) CD34 (+) cells, 20-115 x 10(3) high proliferative potential colony-forming cells (HPP-CFC) and 0.49-2.67 x 10(6) granulocyte-macrophage colony-forming unit (GM-CFU) which constituted 24 +/- 8% (10-36; n = 8) of the total GM-CFU po pulation harvested. Similar, but more variable recoveries of GM-CFU were ob tained from the haematon fractions from patients with breast cancer (21 +/- 13%; n=10), Hodgkin's disease (33 +/- 19%; n=4), non-Hodgkin's lymphoma (2 1 +/- 18; n=7), but the recovery was lower from patients with acute myeloge nous leukaemia (AML) (13 +/- 13%; II = 6), The haematon fraction was enrich ed in CD34(+) cells (25-fold), long-term culture initiating cells (LTC-IC/C AFC, week 5) (3.5-fold), HPP-CFC (2.8-fold) and GM-CFU (2.3-fold) over the huffy coat. Purified CD34(+) cells expanded exponentially and produced 800 to 4000-fold more nucleated cells, 300 to 3500-fold more GIM-CFU and 10 to 80-fold more HPP-CFC in stroma-free suspension culture with interleukin-l ( IL-1 beta), IL-3, IL-6, GM-CSF and stem cell factor (SCF), than did the sta rting cell input. The haematon fraction produced significantly more progeni tor cells than the buffy coat in long-term liquid culture (LTC), This was d ue 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 hae maton includes the most productive haematogenous compartment in human BM. T his simple enrichment strategy, using filter-retained haematons, provides a rational source of BM cells for large scale experimental and/or clinical s tudies on haemopoietic stem cells and on critical accessory stromal cells.