Methods for the Identification, Characterization and Isolation of Adult Stem Cells

• Published in: Tissue engineering Vol. 13; no. 4; p. 915
• Main Authors: Thude, S, Koch, S, Ruehl, M, Mertsching, H
• Format: Journal Article
• Language: English
• Published: 01.04.2007
• ISSN: 1076-3279

Background and Aims: Regenerative medicine aims at the development of biologically active, cellular transplants which compensate for lost organ functions and can stimulate self-regeneration of damaged tissues. Adult stem cells have a high potential for a new generation of cell transplants. Firstly, cells are characterized by specific surface markers and further tested for their differentiation potential. To identify, characterize and isolate the cell populations of interest, antibody-based methods like flow cytom-etry and FACS sorting are used. One limiting factor is the frequency of the target cells in the present population; on the other hand, cell surface markers had to be established. The 'side population' (SP), a subpopulation of hematopoietic stem cells, can be identified and sorted, surface marker-independently, via Hoechst 33342 efflux assay on a FACS Vantage SE equipped with a UV laser. Methods: Epidermal cells were isolated from human biopsy material from circumcisions. The cells were double-stained for the cell surface markers CD49f and CD71 and analyzed on a FACS Calibur. For further characterization an additional 7-AAD cell cycle analysis was set up. The method spectrum was enriched by Hoechst 33342 efflux studies, facilitating the detection and sorting ofSP-cells. Results: The two-colour analysis for the cell surface markers CD49f and CD71 resolved two discrete populations within the epidermal cell preparation. This indicates the existence of different stages of epidermal cell differentiation. For the identification of epidermal stem cells we carried out the Hoechst 33342 efflux assay, but no side population was detected in the isolated epidermal cell population. Conclusions: The difficulty in isolating adult stem cells of different tissues lies in the identification of these cells, which is restricted due to the necessity of established markers. The possibility to detect and isolate stem cells marker independently can be carried out through a combination of microscope technology and a cell sorting unit which sorts according to their phase contrast properties and with cell size. By measuring a multitude of different cells, it will be possible to work out minor differences in these parameters. In addition, raman spectroscopic analysis of cells can be used to identify adult stem cells without antibody-labeling techniques.