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Abstrakt
Preconditioning of Human Adipose-derived Stromal/Stem Cells: Evaluation of Short-term Preincubation Regimens to Enhance their Regenerative Potential
Patrick C Baer, Jürgen M Overath, Anja Urbschat, Ralf Schubert and Helmut Geiger
Objective: Stem cell-based therapy seems to be a promising option to support regeneration after organ failure and tissue injury. The transplantation of MSCs requires cells with a maximum regenerative capacity. Therefore, the research on new strategies to improve the release of regenerative factors of MSCs is urgently needed.
Methods: Human adipose-derived stromal/stem cells (ASC) were isolated from lipoaspirates, characterized, and cultured. Cells were either cultured under standard conditions or preconditioned by incubation in a hypoxic environment (0.5% O2) or in normoxia in the presence of recombinant human tumor necrosis factor-α (TNFα) or recombinant human epidermal growth factor (EGF) for 48 hours. First, seven selected regeneration promoting factors were evaluated by qPCR analysis. Afterwards the secretome of ASCs was estimated using a commercially available protein array for 507 proteins.
Results: PCR analysis showed a differential induction of ASCs` gene expression by the three pretreatments. Whereas ASCs in hypoxia showed a significant mRNA induction of VEGF, FGF-7, and IGF-II, the other pretreatments induced no significant change in VEGF expression. The gene expression of HB-EGF and M-CSF was significantly induced in hypoxia and by incubation with TNFα, but not EGF. Angiopoietin-like 1 mRNA was not significantly induced by all three preconditioning regimens. Evaluation by the protein array revealed that from the 507 proteins investigated 21.9% were found to be more than five-fold increased after incubation in hypoxia (111 of 507 proteins). Preincubation with EGF resulted in an upregulation of 32.3% (164/507), whereas TNFα upregulated 28.8% of all proteins evaluated (146/507).
Conclusion: The findings indicate that all three preconditioning regimens induced a wide variety of proteins. However, short-term pretreatment with EGF induced the highest quantity of proteins, and, therefore, appears to be the best preconditioning regime for cell therapeutic approaches.