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Updated: Aug 19, 2026
Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy
Published on: August 10, 2018
Generation of purified stromal cell cultures that support lymphoid and myeloid precursors
Abstract:
Treatment of Dexter-type long-term bone marrow cultures with the antibiotic mycophenolic acid (MPA) eliminates all hemopoietic cells from the cultures, while a morphologically intact, adherent stromal cell layer is retained. The ability of these MPA treated stromal cell cultures to support long-term hemopoiesis was tested by seeding them with fresh bone marrow cells that had been passed through nylon wool. This procedure yields a relatively stromal cell depleted population of hemopoietic cells. An aliquot of 5 X 10(5) or 2.5 X 10(5) nylon wool passed bone marrow cells bearing the T6 chromosomal marker was seeded onto replicate MPA-treated stromal cell layers. The stromal cells stimulated the proliferation of the bone marrow cells, and nonadherent cells were present for up to 8 weeks of culture. Progenitors of granulocytes and macrophages (CFU-GM) were also present for this period of time despite weekly demi-depopulation, during culture feeding. Karyotypic analysis confirmed that the CFU-GM were derived from the reseeded population. Nylon wool-passed bone marrow cells seeded alone into empty flasks under identical conditions did not survive past 1 week. Cells from the reseeded cultures were also tested for early myeloid precursors (CFU-S) and injected into immunodeficient CBA/N mice to test for the presence of primitive B cell precursors. CFU-S were present in mice killed 11 days following injection of cells, and high levels of B cell colony-forming units (CFU-B) were present in mice 4 weeks post reconstitution. Further studies demonstrated that factors present in medium conditioned by the stromal cells could support the growth of CFU-GM. These data indicate that treatment of long-term bone marrow cultures with MPA results in a population of functional stromal cells.
Insights
Mycophenolic acid (MPA) treatment of bone marrow cultures retains functional stromal cells. These stromal cells effectively support the long-term proliferation and differentiation of hemopoietic stem cells, including myeloid and B cell precursors.
Area of Science:
- Hematology
- Cell Biology
- Stem Cell Research
Background:
- Long-term bone marrow cultures (Dexter-type) are crucial for studying hematopoiesis.
- Hemopoietic cells are typically lost in standard culture conditions.
- Stromal cells form the supportive microenvironment for hemopoiesis.
Purpose of the Study:
- To determine if mycophenolic acid (MPA) treatment yields functional stromal cells for supporting hematopoiesis.
- To assess the capacity of MPA-treated stromal layers to support hemopoietic cell proliferation and differentiation.
- To evaluate the potential of these stromal cells to support primitive myeloid and B cell precursors.
Main Methods:
- Dexter-type long-term bone marrow cultures were treated with mycophenolic acid (MPA) to eliminate hemopoietic cells.
- MPA-treated stromal layers were reseeded with nylon wool-passed bone marrow cells.
- Proliferation and survival of reseeded cells were monitored over 8 weeks.
- Karyotypic analysis confirmed the origin of progeny cells.
- Functional assays included colony-forming unit assays (CFU-GM, CFU-S, CFU-B) and in vivo reconstitution studies in immunodeficient mice.
Main Results:
- MPA treatment effectively eliminated hemopoietic cells, preserving an intact stromal layer.
- MPA-treated stromal layers supported the proliferation and survival of reseeded hemopoietic cells for up to 8 weeks.
- Colony-forming assays confirmed the presence of granulocyte-macrophage progenitors (CFU-GM), early myeloid precursors (CFU-S), and primitive B cell precursors (CFU-B).
- Reseeded cells cultured alone survived for only 1 week, highlighting the supportive role of stromal cells.
- Conditioned medium from stromal cells supported CFU-GM growth, indicating the release of supportive factors.
Conclusions:
- Treatment of long-term bone marrow cultures with MPA generates a population of functional stromal cells.
- These functional stromal cells can support long-term hematopoiesis, including the maintenance of various progenitor populations.
- MPA-treated stromal cell cultures represent a valuable tool for studying the hemopoietic microenvironment and stem cell biology.
