Telomere shortening after ex vivo expansion of tissue-specific stem cells

Konference: 2010 6. sympózium a workshop molekulární patologie a histo-cyto-chemie

Kategorie: Nádorová biologie/imunologie/genetika a buněčná terapie

Téma: Keynote lectures of invited speakers

Číslo abstraktu: 008

Autoři: Prof.MUDr. Jaroslav Mokrý, Ph.D.; MUDr. Tomáš Soukup (1980-), Ph.D.; Doc.MUDr. Stanislav Mičuda, Ph.D.; Mgr. Jan Bouchal; MUDr. Jana Karbanová, Ph.D.; J. Suchánek; MUDr. Benjamin Víšek; E. Brčáková; PharmDr. Doris Vokurková, Ph.D.; R. Ivančáková

In adult multicellular organisms, population of tissue-specific stem cells is responsible for vital functions as tissue maintenance and tissue regeneration. To perform these functions for the entire lifespan, i.e. for more than seven decades in humans, stem cells have to be long living. Of many factors contributing to long-living functionality of tissue-specific stem cells, we focused on measurement of telomere length. After isolation of stem cells, these must be thoroughly characterized to confirm their unique biological properties and expression of stem cell markers.

For analysis, we utilized tissue-specific stem cells isolated from the dental pulp. To expand an initially small number of cells in vitro, stem cells were regularly passaged. We characterized the growth of stem cell lines by measurements of population doublings and doubling times. The cell viability, karyotype and phenotype were checked regularly. Telomere dynamics was examined with quantitative real-time PCR and results were verified by measurement of terminal restriction fragment length and flow FISH.

Dental pulp stem cells (DPSCs) expressed mesenchymal markers CD29, CD44, CD73, CD90, CD166, vimentin and STRO-1 at high levels while hemopoietic markers were negative (CD34, CD45). The cells expressed stem cell markers nanog, SOX-2, nestin, musashi-1, nucleostemin, VEGFR2 and CXCR4. DPSCs are multipotent as shown by their osteogenic and chondrogenic potential. Although these cells have a large proliferative capacity, they show a decline in their doubling time with prolonged cultivation. For the first 42 population doublings, doubling time was 27±6 hrs, however, at later passages doubling time slowed down to 47±7 hrs. All DPSC lines were able to grow beyond Hayflick´s limit. The largest population doubling number reached in our lines was 81. Although measurement of telomere length revealed interindividual differences between the patients, we observed telomere attrition when samples of the same patients from different passages were compared. Comparison of telomere length between DPSCs and lymphocytes from the same patient showed that the latter had shorter telomeres than adult tissue-specific stem cells.

Our results document that ex vivo expansion contributes to telomere shortening which explains a retarded growth of DPSCs at later passages. Similar preliminary results were confirmed by measurement of telomere length in bone marrow mesenchymal stem cells and periodontal ligament stem cells. Although telomere loss in tissue-specific stem cells contributes to a tissue ageing, telomere length in stem cells still remained longer when compared with other somatic cells which likely contributes to extended lifespan of these unique cells.

Supported by MSM 0021620820, MSM 6198959216 and FP7 project 223298 -PurStem.

Datum přednesení příspěvku: 23. 4. 2010