Reversible commitment to differentiation by human multipotent stromal cells in single-cell-derived colonies

Objective: Human multipotent stromal cells readily form single-cell-derived colonies when plated at clonal densities. However, the colonies are heterogeneous because cells from a colony form new colonies that vary in size and differentiation potential when replated at clonal densities. The experiments here tested the hypothesis that cells in the inner regions of colonies are partially differentiated, but the differentiation is reversible. Materials and Methods: Cells were separately isolated from the dense inner (IN) regions and less-dense outer regions (OUT) of single-cell-derived colonies. Cells were then compared by assays of their transcriptomes and proteins, and for clonogenicity and differentiation. Results: IN cells expressed fewer cell-cycle genes and higher levels of genes for extracellular matrix than the OUT cells. When transferred to differentiation medium, differentiation of the colonies occurred primarily in the IN regions. However, the IN cells were indistinguishable from OUT cells when replated at clonal densities and assayed for rates of propagation and clonogenicity. Also, colonies formed by IN cells were similar to colonies formed by OUT cells because they had distinct IN and OUT regions. Cultures of IN and OUT cells remained indistinguishable through multiple passages (30 to 75 population doublings), and both cells formed colonies that were looser and less dense as they were expanded. Conclusions: The results demonstrated that cells in the IN region of single-cell-derived colonies are partially differentiated, but the differentiation can be reversed by replating the cells at clonal densities.