title: The assessment of CD146-based cell sorting and telomere length analysis for establishing the identity of mesenchymal stem cells in human umbilical cord.
abstract: Adult stem cells are characterised by longer telomeres compared to mature cells from the same tissue. In this study, candidate CD146 (+) umbilical cord (UC) mesenchymal stem cells (MSCs) were purified by cell sorting from UC tissue digests and their telomere lengths were measured in comparison to donor-matched CD146-negative fraction. UC tissue fragments were enzymatically treated with collagenase and the cells were used for cell sorting, colony-forming fibroblast (CFU-F) assay or for long-term MSC cultivation. Telomere lengths were measured by qPCR in both culture-expanded MSCs and candidate native UC MSCs. Immunohistochemistry was undertaken to study the topography of CD146 (+) cells. Culture-expanded UC MSCs had a stable expression of CD73, CD90 and CD105, whereas CD146 declined in later passages which correlated with the shortening of telomeres in the same cultures. In five out of seven donors, telomeres in candidate native UC MSCs (CD45 (-)CD235α (-)CD31 (-)CD146 (+)) were longer compared to donor-matched CD146 (-) population (CD45 (-)CD235α (-)CD31 (-)CD146 (-)). The frequency of CD45 (-)CD235α (-)CD31 (-)CD146 (+) cells measured by flow cytometry was ~1000-fold above that of CFU-Fs (means 10.4% and 0.01%, respectively). CD146 (+) cells were also abundant in situ having a broad topography including high levels of positivity in muscle areas in addition to vessels. Although qPCR-based telomere length analysis in sorted populations could be limited in its sensitivity, very high frequency of CD146 (+) cells in UC tissue suggests that CD146 expression alone is unlikely to be sufficient to identify and purify native MSCs from the UC tissue.
authors.affiliation: Department of Biomedical Research, Foundation for Research and Technology-Hellas, Institute of Molecular Biology and Biotechnology, University of Ioannina, Ioannina, 45110, Greece.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Kouroupis
authors.affiliation: Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, LS97TF, UK ; NIHR Leeds Musculoskeletal Biomedical Research Unit, Leeds Teaching Hospitals NHS Trust, Leeds, LS97TF, UK.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Churchman
authors.affiliation: Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, LS97TF, UK ; NIHR Leeds Musculoskeletal Biomedical Research Unit, Leeds Teaching Hospitals NHS Trust, Leeds, LS97TF, UK.
authors.forename: Dennis
authors.initials: D
authors.lastname: McGonagle
authors.affiliation: Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, LS97TF, UK ; NIHR Leeds Musculoskeletal Biomedical Research Unit, Leeds Teaching Hospitals NHS Trust, Leeds, LS97TF, UK.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Jones