Background: Human adipose tissue-derived mesenchymal stromal cells (AD-MSCs) have been under focus

in regenerative medicine since their discovery as a suitable source of MSCs. AD-MSCs are heterogeneous

cells and exhibit variations in population doubling time, morphology and proliferative capacity. This study

investigated if human AD-MSCs are developing, during in vitro long-term cultivation, in an unwanted or

aberrant way.

Methods: This study monitored AD-MSCs during their in vitro culture till the tenth passage investigating

proliferation kinetics, DNA index and surface markers expression. Also, periostin gene expression was


Results: The proliferation capacity and colony forming unit were decreased after passage 6 and the

population doubling time was increased. Flow cytometric analysis revealed that newly cultivated population

strongly expressed MSCs markers, furthermore, reduction of CD105 expression appeared in passage 5

onwards, the later was associated with significant increase in expression of CD34 (a hematopoietic cell

marker). Also, reduction of CD73 and CD90 expression was observed from passage 8. Furthermore, during

the frst six passages, periostin expression was signifcantly unchanged, with signifcant upregulation in late


Conclusions: Long-term cultivation of human AD-MSCs changed their characters in an aberrant

way and the first four passages might be the most appropriate passages for therapy. More investigation

and understanding of these variations are needed to help in standardizing the expansion of MSCs-based


Keywords: AD-MSCs; cell proliferation assay; colony forming unit (CFU); DNA index (DI); periostin gene