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Growth characteristics of PHA-induced colonies in primary and secondary agar culture
Abstract:
PHA-induced colonies were obtained from peripheral blood mononuclear cells (MC) grown in agar-medium. When the colonies were harvested from mass cultures, pooled as single cell suspensions and plated again in presence of PHA, they failed to generate new colonies unless they were seeded on an underlayer containing uncultured blood MC. Cytogenetic studies indicate that most secondary colonies were derived from primary colonies. Autologous as well as heterologous feeder cells were able to promote the growth of secondary colonies. No granulocyte (G) or macrophage (M) colony formation was observed in secondary cultures. These experiments show that the progenitors of PHA-induced colonies differ from G or M CFCs and that they are still detected in these colonies which contain 82 +/- 12% T-cells. In contrast, colony formation requires the presence of factor(s) provided by cooperating cells (CC) which are no longer detected in primary colonies and this is associated with a depletion in non-T elements from the initial MC population.
Insights
PHA-stimulated colonies require cooperating cells for secondary growth. These PHA-induced colonies, rich in T-cells, differ from granulocyte or macrophage progenitors.
Area of Science:
- Immunology
- Cell Biology
Background:
- Peripheral blood mononuclear cells (MC) form colonies when stimulated with phytohemagglutinin (PHA).
- The cellular composition and growth requirements of these PHA-induced colonies are not fully understood.
Purpose of the Study:
- To investigate the cellular requirements for secondary colony formation from PHA-stimulated primary colonies.
- To characterize the progenitor cells within PHA-induced colonies and their relationship to other hematopoietic progenitors.
Main Methods:
- Primary PHA-induced colonies were cultured from peripheral blood mononuclear cells (MC).
- Secondary cultures were established using cells from primary colonies, with and without feeder layers of uncultured MC.
- Cytogenetic analysis and assessment of granulocyte (G) or macrophage (M) colony formation were performed.
Main Results:
- Secondary colonies failed to form without an underlayer of uncultured MC (feeder cells).
- Cytogenetic studies confirmed most secondary colonies originated from primary colony cells.
- No granulocyte (G) or macrophage (M) colony formation was observed in secondary cultures.
- Primary colonies contained 82% T-cells, but lacked essential cooperating cells (CC) for secondary growth.
Conclusions:
- PHA-induced colony progenitors are distinct from G or M colony-forming cells (CFCs).
- Secondary colony formation necessitates factors from cooperating cells (CC), which are depleted in primary colonies.
- The findings highlight the role of cooperating cells in supporting the proliferation of PHA-responsive T-cell progenitors.