Passage- and serum-dependent changes of adipose-derived stromal cells in vitro: a discrepancy of stemness factors regarding mesenchymal surface markers and expression of stemness-related genes.

Objectives: Autologous fat transplantation is a promising source for cell therapy and tissue engineering. However, the physiological function and regulatory mechanisms of in vitro cell culture remain largely unexplored. Furthermore, no standard protocol for cell culture of human adipose-derived stem cells (hASC) has been described. Previous studies have reported the impact of media supplementation on the loss of stemness capacity. Methods: In this study, we compared the expression of stemness-defining surface markers according to the minimal criteria definition (CD 11b⁻, CD 31⁻, CD 34⁻, CD 45⁻, CD 73⁺, CD 90⁺, and CD 105⁻) by flow cytometry analysis with the expression of stemness-related genes such as MCAM, OCT4, MYC, and cKit in hASCs cultured in either fetal calf serum (FCS) or human serum (HS) supplemented medium from passage 0 to 5. Results: As expected, we found that hASCs in both groups retained their typical mesenchymal surface marker profile CD 73 and CD 90 (>95 %) in flow cytometry analysis, as well as the absence of CD 11b, CD 31, CD 34, and CD 45 (<5 %) until passage 5. However, in contrast to that, RT-PCR indicated a passage-dependent decline and medium-dependent changes in the transcriptome, in particular the loss of the stemness-related genes cKit and MCAM in both groups, while MYC and OCT4 showed unpredictable expression. Conclusions: Summarized, these results indicate the need for standardized cell culture protocol, as the transcriptome seems to change during in vitro cultivation, although an ASC-typical pattern of surface markers remains. In this regard, our study aims to contribute to the establishment of a standard protocol to achieve reliability, validity, and objectivity for future cell therapy or clinical applications.