Discrete adipose-derived stem cell subpopulations may display differential functionality after in vitro expansion despite convergence to a common phenotype distribution

Background: Complex immunophenotypic repertoires defining discrete adipose-derived stem cell (ASC) subpopulations may hold a key toward identifying predictors of clinical utility. To this end, we sorted out of the freshly established ASCs four subpopulations (SPs) according to a specific pattern of co-expression of six surface markers, the CD34, CD73, CD90, CD105, CD146, and CD271, using polychromatic flow cytometry. Method: Using flow cytometry-associated cell sorting and analysis, gating parameters were set to select for a CD73(+)CD90(+)CD105(+) phenotype plus one of the four following combinations, CD34(-)CD146(-)CD271(-)(SP1), CD34(-)CD146(+)CD271(-)(SP2), CD34(+)CD146(+)CD271(-)(SP3), and CD34(-)CD146(+)CD271(+) (SP4). The SPs were expanded 700- to 1000-fold, and their surface repertoire, trilineage differentiation, and clonogenic potential, and the capacity to support wound healing were assayed. Results: Upon culturing, the co-expression of major epitopes, the CD73, CD90, and CD105 was maintained, while regarding the minor markers, all SPs reverted to resemble the pre-sorted population with CD34(-)CD146(-)CD271(-) and CD34(-)CD146(+)CD271(-) representing the most prevalent combinations, followed by less frequent CD34(+)CD146(-)CD271(-) and CD34(+)CD146(+)CD271(-) variants. There was no difference in the efficiency of adipo-, osteo-, or chondrogenesis by cytochemistry and real-time RT-PCR or the CFU capacity between the individual SPs, however, the SP2(CD73+90+105+34-146+271-) outperformed others in terms of wound healing. Conclusions: Our study shows that ASCs upon culturing inherently maintain a stable distribution of immunophenotype variants, which may potentially disguise specific functional properties of particular downstream lines. Furthermore, the outlined approach suggests a paradigm whereby discrete subpopulations could be identified to provide for a therapeutically most relevant cell product.