Bento 2017 MITOEAGLE Obergurgl
Event: MitoEAGLE Obergurgl 2017
Human urine contains a small population of cells with stemness properties, urine-derived stem cells (USCs). USCs can be non-invasively collected and used for personalized therapeutics. However, only a small number of studies about USCs is available. Thus, a deeper characterization of these cells is essential, including in what regards to their mitochondrial function profile. Our aim is to establish a protocol to isolate and expand USCs, enabling for a mitochondrial metabolic characterization.
Urine from female volunteers was used to isolate and expand USCs using the commonly used mixture of Keratinocyte Serum Free Medium and Embryonic Fibroblast Medium (KSFM+EFM) and a mixture of KSFM and DMEM, 10%FBS, 1% MEM-NEAA (KSFM+DMEM). Isolated cells were characterized by flow cytometry for mesenchymal stem cells markers (CD44, CD73, CD90 and CD105) and hematopoietic stem cells markers (CD14, CD20, CD34 and CD45). Moreover, the renal progenitor/multipotent marker CD24 was also assessed. For mitochondrial function profiling, different cell densities were used to determine the oxygen consumption rate (OCR) using the Seahorse XFe96 Extracellular Flux Analyzer. The optimized conditions used to measure OCR involved 20,000 cells/well, with oligomycin 2 μM, FCCP 0.5 μM and rotenone/antimycin A 1 μM used to titrate respiration.
The phenotyping results for USCs isolated from one volunteer using the two media described did not show statistically significant differences. Using the KSFM+DMEM medium, the USCs from six volunteers were ˃96% positive for CD44, CD73 and CD24. The expression of CD90 and CD105 was unstable with average values for positive cells of 60 and 68%, respectively, and the expression of hematopoietic stem cells markers is ˂ 1%. We observed that 77.5% of the OCR was used by USC to sustain ADP phosphorylation and the basal respiration corresponds to 57.2% of their maximal capacity.
In our study, the KSFM+EFM could be replaced by KSFM+DMEM for USCs isolation and expansion from human urine, with similar results regarding stem cells markers. Isolated USCs showed a respiratory reserve capacity which may be used for boosting mitochondrial metabolism after cell differentiation. Future studies will be performed in order to characterize the glycolytic and mitochondrial function profile of USCs.
• Bioblast editor: Kandolf G
Labels: MiParea: Respiration
Organism: Human Tissue;cell: Stem cells Preparation: Intact cells
Coupling state: LEAK, ET Pathway: ROX
Event: A1, Oral
Affiliations and support
- Bento G(1), Oliveira PO(2), Sardão VA(2)
- PhDOC PhD Program, CNC/IBILI, UC-Biotech
- CNC/IBILI, UC-Biotech
- Biocant Park, Univ Coimbra, Portugal. - [email protected]
- This work is supported by FEDER/COMPETE/FCT National Funds–Portugal (PD/BD/114119/2015, PTDC/DTP-FTO/2433/2014, IF/01182/2015 and COMPETE: POCI-01-0145-FEDER-007440).