Human mixed cell colonies: unicellular or multicellular origin--analysis by G-6-PD

Area of Science:

• Hematology
• Stem Cell Biology
• Genetics

Background:

• Accurate enumeration of multipotent stem cells (CFU-mix) is crucial for understanding hematopoietic differentiation.
• Previous studies have not rigorously defined optimal plating conditions for CFU-mix assays.
• X-chromosome-linked glucose-6-phosphate dehydrogenase (G-6-PD) heterozygosity provides a useful marker for clonal analysis.

Purpose of the Study:

• To determine the optimal cell concentrations for reliable enumeration of multipotent stem cells (CFU-mix) using an in vitro colony assay.
• To validate the accuracy of CFU-mix enumeration under varying cell plating densities.
• To assess the impact of cell concentration on the reliability of hematopoietic stem cell assays.

Main Methods:

• Culturing marrow and peripheral blood cells from G-6-PD heterozygous women at various cell concentrations (2 X 10(4)/ml to 4 X 10(5)/ml).
• Utilizing velocity sedimentation to isolate cells prior to plating.
• Determining the G-6-PD enzyme type of individual colonies to identify monoclonally derived versus multi-cellular colonies.

Main Results:

• At cell concentrations ≤7.5 X 10(4)/ml for marrow and ≤1.25 X 10(5)/ml for peripheral blood, colonies exhibited a single G-6-PD enzyme type (A or B).
• At higher cell concentrations, a significant proportion of colonies displayed both G-6-PD enzyme types, indicating they originated from more than one cell.
• These findings demonstrate that CFU-mix enumeration is accurate only at low cell densities.

Conclusions:

• In vitro colony assays for CFU-mix are only accurate when performed at low cell concentrations.
• Studies investigating hematopoietic differentiation using CFU-mix assays must account for cell concentration effects.
• The reliability of multipotent stem cell enumeration is highly dependent on adherence to specific plating density guidelines.