Hematopoietic stem cells are extremely difficult to maintain or expand
in vitro. Two observations in traditional long-term
bone marrow cultures strongly suggest that macrophages may be at the root of the problem: First, micromolar concentrations of
hydrocortisone improve the
longevity of long-term
bone marrow cultures and
hydrocortisone is known as a potent inhibitor of macrophage production of pro-inflammatory cytokines, chemokines, enzymes,
nitrogen oxide and
reactive oxygen species and redirects macrophages to the anti-inflammatory differentiation pathway; Second, the decline of hematopoiesis in long-term
bone marrow cultures coincides with the development of large numbers of adherent and non-adherent macrophages including
foreign body giant cells. These adherent macrophages and
foreign body giant cells exhibit well-spread morphology, contain numerous lysosomes and phagolysosomes in the
cytoplasm and are metabolically active. We hypothesize that
hydrocortisone fails to suppress all aspects of macrophage pro-inflammatory activation / differentiation, resulting in the production of inhibitors or toxins of hematopoiesis. Macrophage adhesion in
cell culture depends on serum proteins pre-adsorbed to the tissue-culture-treated
polystyrene (TC-PS), which adsorbs proteins via mostly
hydrophilic interactions. TC-PS is used in almost all
tissue culture devices currently available. Cellular adhesion provides a strong stimulus for metabolic, mitotic and certain
gene activities. Therefore, we seek to reduce macrophage adhesion and activation by culturing bone marrow cells in
tissue culture devices composed of or covered with polymers with very different
protein-binding characteristics than TC-PS such as
polyethylene (PE) and other polyolefins, the latter bind proteins via exclusively hydrophobic interactions. As a result, polyolefins bind different proteins and in lower quantities than TC-PS. Furthermore, PE does not contain additional chemical features like the phenolic rings of
polystyrene that might contribute to
protein binding and macrophage adhesion / activation. Using these new culture devices, we developed a drastically different long-term bone marrow culture, the “Low Macrophage-Adhesion / Activation” (LoMAC) bone marrow culture. In LoMAC bone marrow culture, hematopoiesis continues for months to over a year and hematopoietic stem cells are amplified gradually. In stark contrast to traditional long-term bone marrow cultures, de novo
erythropoiesis and megakaryocytopoiesis proceed robustly in the LoMAC bone marrow culture and B-
lymphocyte and
natural killer cell progenitors can be continuously derived. Thus, these new culture devices and the associated LoMAC culture method offer a new way to study hematopoiesis
in vitro and provide a more robust platform for the expansion of hematopoietic stem cells and progenitors
ex vivo.