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Updated: Sep 21, 2026
Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture
Published on: May 8, 2018
Clonal nature of pluripotent hemopoietic precursors in vitro (CFU-mix)
Abstract:
Direct evidence is presented for the clonal nature of the mixed colonies from pluripotent hemopoietic precursors in vitro in adult murine marrow cells. Bone marrow cells from BDF1 mice were fractionated using the technique of velocity sedimentation according to Miller and Phillips to enrich the concentration of the mixed colony-forming units. After 3 h sedimentation, marrow cells in the 15th fraction were harvested and cultured using a methylcellulose culture assay. After 2 days of culture, dividing single cells were individually aspirated and transferred to fresh culture medium for mixed colonies using a Leitz micromanipulator. 92 macrophage-granulocyte colonies, 11 erythropoietic bursts, and 4 mixed colonies were detected in 358 single-cell culture experiments. The present results indicate that mixed colonies are clonal, and the mixed colony-forming units are pluripotent.
Insights
This study provides direct evidence that mixed colonies derived from adult mouse bone marrow cells are clonal. The findings confirm that mixed colony-forming units possess pluripotent hemopoietic stem cell properties.
Area of Science:
- Hematology
- Stem Cell Biology
- Cell Biology
Background:
- Understanding the nature of hemopoietic stem cells is crucial for regenerative medicine.
- Previous studies suggested heterogeneity in colony formation from bone marrow cells.
Purpose of the Study:
- To investigate the clonal origin of mixed colonies formed in vitro from murine bone marrow.
- To determine if mixed colony-forming units are pluripotent hemopoietic precursors.
Main Methods:
- Fractionation of adult BDF1 mouse bone marrow cells using velocity sedimentation.
- Culture of enriched fraction cells in methylcellulose assay.
- Single-cell transfer and culture using a micromanipulator to assess colony formation.
Main Results:
- Analysis of 358 single-cell cultures revealed 92 macrophage-granulocyte colonies, 11 erythropoietic bursts, and 4 mixed colonies.
- The formation of diverse cell types within single colonies supports a clonal origin.
- The results demonstrate that mixed colonies arise from single, multipotent progenitor cells.
Conclusions:
- Mixed colonies observed in vitro are indeed clonal in nature.
- Mixed colony-forming units represent pluripotent hemopoietic stem cells capable of differentiating into multiple lineages.
