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Lymphohematopoietic progenitors of normal mice
T C Ball1, F Hirayama, M Ogawa
1Department of Medicine, Medical University of South Carolina, Charleston, USA.
Insights
Researchers identified lymphohematopoietic progenitors in mouse bone marrow. Flow cytometry improved detection of B-cell components within these crucial stem cells.
Area of Science:
- Hematology
- Stem Cell Biology
- Immunology
Background:
- Identifying and characterizing lymphohematopoietic progenitors is crucial for understanding blood cell development.
- Previous methods for identifying these progenitors had limitations in accuracy and efficiency.
Purpose of the Study:
- To identify and characterize lymphohematopoietic progenitors in normal mouse bone marrow.
- To compare different methods for detecting lymphomyeloid colonies.
Main Methods:
- Single-step methylcellulose culture assay using lineage-negative Ly-6A/E (Sca-1)+ progenitors.
- Culture supplemented with steel factor (SF), interleukin-7 (IL-7), erythropoietin (Ep), and IL-11.
- Flow-cytometric analysis of B220-positive cells for colony identification, confirmed by micromanipulation.
Main Results:
- Flow cytometry proved superior to microscopy for identifying B220-positive cells in lymphomyeloid colonies.
- SF, IL-7, and Ep supported lymphomyeloid colony formation; IL-11, G-CSF, or IL-12 enhanced it.
- IL-1 alpha and IL-3 inhibited B-lymphoid lineage expression.
- Normal mice possess approximately four times more lymphohematopoietic progenitors than 5-fluorouracil treated mice.
Conclusions:
- Flow cytometry is a more effective method for identifying lymphomyeloid colonies.
- Specific cytokines influence the development and number of lymphohematopoietic progenitors.
- Normal bone marrow contains a significantly higher number of these progenitors compared to chemotherapy-treated bone marrow.
Abstract:
We have identified and characterized the lymphohematopoietic progenitors in the bone marrow of normal mice using a single-step methylcellulose culture assay. Lineage-negative Ly-6A/E (Sca-1)+ progenitors isolated from normal mice were plated in methylcellulose culture containing steel factor (SF), interleukin-7 (IL-7), erythropoietin (Ep), and IL-11. After 16 to 17 days of culture, pre-B-cell-containing multilineage myeloid colonies can be microscopically identified; however, flow-cytometric analysis of individual colonies for B220-positive cells proved superior to in situ microscopic identification of lymphomyeloid colonies. Approximately 10% (6/66) of the mixed colonies without a conspicuous B-cell component had B220-positive cells. The single cell origin of the lymphomyeloid colonies was confirmed by micromanipulation. Although the combination of SF, IL-7, and Ep was sufficient to support formation of lymphomyeloid colonies, addition of IL-11, granulocyte colony-stimulating factor or IL-12 to the combination of SF, IL-7, and Ep increased the number of lymphomyeloid colonies. IL-1 alpha and IL-3 independently inhibited the expression of the B-lymphoid lineage when added to the combination of SF, IL-7, Ep, and IL-11. Approximately four times more lymphohematopoietic progenitors are present in normal mice than in mice treated with 5-fluorouracil.