Hematopoiesis and Stem Cell Renewal in Long-Term Bone Marrow Cultures Containing Catalase.

Many of the events that occur within the bone marrow can be modeled in long-term bone marrow cultures (LTBMC), which are capable of producing stem cells. Although the cultures faithfully replicate the differentiation of many hematopoietic lineages, they are relatively short-lived. The stem cell compartment is rapidly depleted and attempts to achieve expansion of hematopoietic cells in culture have met with limited success. These cultures accumulate large numbers of granulocytes and monocytes capable of producing significant levels of reactive oxygen species (ROS). It has recently become clear that some ROS, including H₂O₂ can play a critical role in intracellular signalling induced by various growth factors and cytokines. We therefore elected to test the effect of 2 different H₂O₂ scavenger catalases, (bovine or aspergillosis added on alternate days) on LTBMC hematopoiesis of mouse low density bone marrow cells on irradiated adherent preformed stromal monolayers. Dramatic alterations were noted with either catalase, whereas heat-inactivated catalase had no effect. Initially there is a 5–10 fold increase in the non-adherent granulocytes and their precursors. The increase is relatively short-lived at 3–4 weeks when catalase cultures contain 1/5 as many hematopoietic cells as controls. However these cells contain 5 times the number of myeloid clonal progenitors (CFU-c) than controls. After 4–5 weeks the catalase treated cells become quiescent. When catalase is removed hematopoiesis returns promptly, ruling out a catalase-induced toxic effect. By the 3^rd week of catalase treatment >90% of non-adherent cells are Sca-1⁺ and 36% of them are Lin⁻. In absolute numbers the Sca-1⁺ and Lin⁻ population increase 80 fold at 3 weeks. If losses induced by removal of half of the non-adherent cells with each weekly feeding are considered, the absolute increase is >500 fold. Virtually all of the Sca-1⁺, Lin⁻ cells express C-Kit⁺. At 2–3 weeks, approximately 15% of cells recovered from the catalase cultures have this stem cell phenotype described for murine cells, which represents a 200 fold increase in stem cells compared to controls. These cells (20,000 Ly 5.1 cells) were then tested for their ability to sustain both short- and long-term hematopoiesis in lethally irradiated Ly 5.2 mice along with 30,000 freshly isolated Ly 5.2 bone marrow cells. The catalase-treated cells showed both short- and long-term repopulating activity. At 3,6 and 10 weeks sorted Sca-1⁺, Lin⁻ catalase-treated Ly 5.1 cells were 14,20 and 39% respectively of host cells, compared to 1,3 and 5% of cells cultured without catalase. These catalase-treated cells underwent multilinege repopulation granulocytes (Gr-1⁺), monocytes (mac-1⁺), T-cells (CD3⁺) and B⁻ cells (B-220⁺) in the Ly 5.2 host. Thus, peroxide-sensitive regulatory mechanisms play an important role in regulating hematopoietic stem cell renewal and differentiation. Protected from H₂O₂, hematopoietic progenitors multiply and become quiescent. These cells are 200–500 fold enriched with functional stem cells. Manipulation of peroxide levels in vitro can dramatically enhance the growth of self-renewing hematopoietic stem cells and may provide a unique source of undifferentiated hematopoietic progenitors.