Kouroupis Dimitrios, Churchman Sarah M, McGonagle Dennis, Jones Elena A
Department of Biomedical Research, Foundation for Research and Technology-Hellas, Institute of Molecular Biology and Biotechnology, University of Ioannina, Ioannina, 45110, Greece.
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.
F1000Res. 2014 Jun 11;3:126. doi: 10.12688/f1000research.4260.2. eCollection 2014.
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.
与来自同一组织的成熟细胞相比，成体干细胞的特征在于端粒更长。在本研究中，通过细胞分选从脐带（UC）组织消化物中纯化候选CD146（+）间充质干细胞（MSC），并与供体匹配的CD146阴性部分比较测量其端粒长度。UC组织片段用胶原酶进行酶处理，细胞用于细胞分选、集落形成成纤维细胞（CFU-F）测定或长期MSC培养。通过qPCR在培养扩增的MSC和候选天然UC MSC中测量端粒长度。进行免疫组织化学以研究CD146（+）细胞的拓扑结构。培养扩增的UC MSC具有CD73、CD90和CD105的稳定表达，而CD146在后期传代中下降，这与同一培养物中端粒的缩短相关。在七个供体中的五个中，候选天然UC MSC（CD45（-）CD235α（-）CD31（-）CD146（+））中的端粒比供体匹配的CD146（-）群体（CD45（-）CD235α（-）CD31（-）CD146（-））更长。通过流式细胞术测量的CD45（-）CD235α（-）CD31（-）CD146（+）细胞频率比CFU-F高约1000倍（分别为10.4%和0.01%）。CD146（+）细胞在原位也很丰富，具有广泛的拓扑结构，除血管外，在肌肉区域也有高水平的阳性表达。尽管基于qPCR的分选群体中端粒长度分析的敏感性可能有限，但UC组织中CD146（+）细胞的非常高频率表明，仅CD146表达不太可能足以从UC组织中鉴定和纯化天然MSC。
