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

Abstract

Summary: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 × 10 6 CD34 + cells, 20-115 × 10 3 high proliferative potential colony-forming cells (HPP-CFC) and 0.49-2.67 × 10 6 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-1␤), 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,

Mesenchymal Stem Cells Derived from CD133‐Positive Cells in Mobilized Peripheral Blood and Cord Blood: Proliferation, Oct4 Expression, and Plasticity

“…We observed a great variability between BM samples (BM1 versus BM2). BM1 contained more MSCs than BM2, but BM1 corresponds to particulate fraction removed by nylon filtration prior to transplantation, which is rich in hematopoietic and stromal cells [34]. BM2 is obtained though classic sternal aspiration.…”

Fibroblast Growth Factor-2 Enhances Proliferation and Delays Loss of Chondrogenic Potential in Human Adult Bone-Marrow-Derived Mesenchymal Stem Cells

“…3 In some cases, MSCs can be expanded through 30-40 population doublings (10 9 -10 12 :1 expansion), whereas other preparations stop proliferating after 4-5 population doublings (50:1 expansion). [4][5][6] This different behavior may be because of multiple factors, such as the protocol used to obtain the bone marrow, [4][5][6][7] the low frequency of MSCs in marrow, 8 and the age and condition of the donor. 5,9 Interestingly, the loss of differentiation potential is not generalized.…”

Progenitor cells trapped in marrow filters can reduce GvHD and transplant mortality

“…Our findings are in keeping with studies showing that stromal cells and haemopoietic progenitors form solid complexes (haematons) that are trapped in BM filters. 6,7 In a study performed by Blasek, 12 there were 3.5 times more LTC-ICs in the haematon particles extracted from filters than in the buffy coats of filtered human BM isolates. The authors also studied the association of HPP-CFC and GM-CFU with stromal cells by determining the frequencies of CFU-F in buffy coat and haematon fractions: only in the latter, HPP-CFC generated CFU-F in long-term culture.…”